Btla antibodies

ABSTRACT

This invention relates generally to antibodies or antigen binding fragments that bind to human B and T lymphocyte attenuator (BTLA) and uses thereof. More specifically, the invention relates to agonistic antibodies that bind human BTLA and modulate its activity, and their use in treating inflammatory, autoimmune and proliferative diseases and disorders.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.17/330,168, filed May 25, 2021, which is a continuation of InternationalApplication No. PCT/GB2019/053569, filed Dec. 17, 2019, which claims thebenefit of United Kingdom Application No. 1820554.2, filed 17 Dec. 2018,each of which is incorporated by reference in its entirety for allpurposes.

REFERENCE TO A SEQUENCE LISTING

The Instant application contains a Sequence Listing which has beensubmitted is ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 27, 2021, isnamed 56270_708_302_SL.txt and is 104,908 bytes in size.

FIELD OF THE INVENTION

This invention relates generally to antibodies or antigen bindingfragments that bind to human B and T lymphocyte attenuator (BTLA) anduses thereof. More specifically, the invention relates to agonisticantibodies that bind human BTLA and modulate its activity, and their usein treating inflammatory, autoimmune and proliferative diseases anddisorders.

BACKGROUND

The immune system must achieve a balance between the destruction ofpathogens or dangerously mutated cells and tolerance of healthyself-tissue and innocuous commensals. To facilitate this balance theactivity of immune cells is influenced by the integration of signalsfrom multiple stimulatory and inhibitory receptors that attune cells totheir environment. These surface-expressed receptors present attractivetargets for the therapeutic modulation of immune responses. Many humandiseases result from aberrant or unwanted activation of the immunesystem including autoimmune diseases, transplant rejection andgraft-versus-host disease. Agonist agents capable of inducing signalingthrough inhibitory receptors could dampen these unwanted immuneresponses. B and T lymphocyte attenuator (BTLA; also designated CD272)is an inhibitory member of the CD28 family of receptors that alsoincludes CD28, CTLA-4, ICOS, and PD-1 (Watanabe et al., Nat Immunol.4:670-679, 2003) It is widely expressed throughout the immune system onboth myeloid and lymphoid cells (Han et al., J Immunol. 172:5931-9,2004). Following engagement by its ligand herpesvirus entry mediator(HVEM), BTLA recruits the phosphatases SHP-1 and SHP-2 to itscytoplasmic domain (Sedy et al., Nat Immunol. 6:90-8, 2005), which inturn inhibit the signaling cascades of activating receptors. Micelacking an intact BTLA gene show hyperproliferative B and T cellresponses in vitro, higher titers to DNP-KLH post-immunization and anincreased sensitivity to EAE (Watanabe et al, Nat. Immunol, 4:670-679,2003). If observed until old age BTLA knock-out mice spontaneouslydevelop autoantibodies, an auto-immune hepatitis like disease andinflammatory cell infiltrates in multiple organs (Oya et al., ArthritisRheum 58: 2498-2510, 2008). This evidence indicates that the BTLAinhibitory receptor plays a crucial role in maintaining immunehomeostasis and inhibiting autoimmunity. Furthermore, HVEM-BTLAsignaling is involved in the regulation of mucosal inflammation andinfection immunity (Shui et al., J Leukoc Biol. 89:517-523, 2011).

Therapeutic agents that are capable of modulating BTLA function toinhibit autoreactive lymphocytes in the context of autoimmune disorderswould be highly desirable.

It has previously been shown that monoclonal antibodies binding to mouseBTLA can act as agonists, inducing signaling through the receptor toinhibit immune cell responses. In the presence of agonist anti-BTLAantibody (mAb), anti-CD3 and anti-CD28 activated T-cells show reducedIL-2 production and proliferation (Kreig et al., J. Immunol., 175,6420-6472, 2005).

Furthermore, anti-mouse-BTLA agonist antibodies have been shown toameliorate disease in murine models of graft-versus-host disease (Sakodaet al., Blood. 117:2506-2514; Albring et al., J Exp Med. 207:2551-9,2010). Agonist antibodies targeting the human BTLA receptor have beenshown to inhibit T cell responses ex-vivo (see Otsuki et al., BiochemBiophys Res Commun 344:1121-7, 2006; and WO2011/014438), but have notyet been translated to the clinic.

There is a need in the art to discover new and useful agents, such asantibodies or antigen-binding antibody fragments, capable of modulatingBTLA.

BRIEF SUMMARY OF THE DISCLOSURE

The present invention relates to anti-human BTLA agonist antibodies orantibody fragments thereof having one or more desirable properties,including high binding affinities, high agonist potency, high agonistefficacy, good pharmacokinetics and low antigenicity in human subjects.The invention also relates to use of the antibodies or antibodyfragments of the invention in the treatment of disease.

According to a first aspect of the invention there is provided anisolated antibody or an antigen-binding fragment thereof thatspecifically binds BTLA, wherein the antibody has a heavy chain and/orlight chain with at least one complementarity-determining region (CDR)as present in an antibody selected from the group consisting of 11.5.1,2.8.6, 12F11, 14D4, 15B6, 15C6, 16E1, 16F10, 16H2, 1H6, 21C7, 24H7,26B1, 26F3, 27G9, 3A9, 3E8, 4B1, 4D3, 4D5, 4E8, 4H4, 6G8, 7A1, 8B4, 8C4,6.2 and 831, as identified in Table 1 and described herein.

According to a second aspect of the invention there is provided anisolated nucleic acid comprising a nucleotide sequence that encodes aheavy chain polypeptide or a light chain polypeptide of the isolatedantibody or an antigen-binding fragment thereof of the first aspect ofthe invention.

According to a third aspect of the invention there is provided a vectorcomprising the nucleic acid of the second aspect of the invention.

According to a fourth aspect of the invention there is provided a hostcell comprising the nucleic acid sequence according to the second aspectof the invention or the vector according to the third aspect of theinvention.

According to a fifth aspect of the invention there is provided a methodof producing an antibody or an antigen-binding fragment thereofaccording to the first aspect of the invention, comprising the step ofculturing the host cell of the fourth aspect of the invention underconditions for production of said antibody or an antigen-bindingfragment thereof, and optionally isolating and/or purifying saidantibody or an antigen-binding fragment thereof

According to a sixth aspect of the invention there is provided apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of the antibody orantigen-binding fragment thereof of the first aspect of the invention,or that produced by the fifth aspect of the invention.

According to a seventh aspect of the invention there is provided amethod of preparing a pharmaceutical composition, the method comprisingformulating antibody or an antigen-binding fragment thereof inaccordance with the first aspect of the invention, or one produced inaccordance with the fifth aspect of the invention into a compositionincluding at least one additional component. In a particular embodiment,the at least one additional component is a pharmaceutically acceptableexcipient.

According to an eighth aspect of the invention there is provided amethod of treating a BTLA-related disease in a patient, comprisingadministering to the patient a therapeutically effective amount of theantibody or antigen-binding fragment thereof of the first aspect of theinvention or the pharmaceutical composition of the sixth aspect of theinvention.

DETAILED DESCRIPTION

The inventors have identified particularly strong agonist antibodies toBTLA which are predicted to be more efficacious than current antibodiesat suppressing T cell responses and thus be particularly useful in thetreatment of immune mediated disorders.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a molecule”optionally includes a combination of two or more such molecules, and thelike.

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be applied to any aspect unlessthe content clearly dictates otherwise.

Furthermore, that the various embodiments may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art. These andother embodiments of the invention are further described by the detaileddescription that follows.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof ordinary skill with a general dictionary of many of the terms used inthis disclosure.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The numbering of amino acids in the variable domain, CDRs and frameworkregions (FRs), of an antibody follow, unless otherwise indicated, theKabat definition as set forth in Kabat et al. Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, MD. (1991).

The terms “B and T lymphocyte attenuator” and “BTLA” are usedinterchangeably and, unless the context dictates otherwise, withreference to either the protein or gene (or other nucleic acid encodingall or part of BTLA). The human BTLA sequences encompass all humanisotype and variant forms. A representative example of full length humanBTLA is disclosed in Genbank under accession number: AJ717664.1. Anotherrepresentative polypeptide sequence of human BTLA is disclosed in SEQ IDNO: 23, which only differs from that in AJ717664.1 by two naturalvariant single nucleotide polymorphisms. Despite allelic variations, ahuman BTLA polypeptide sequence will typically have at least 90%sequence identity (such as at least 95%, 96%, 97%, 98%, 99% or 100%) tohuman BTLA in SEQ ID NO: 23.

A representative example of full length cynomolgus (cyno) BTLA isdisclosed in Genbank under accession number: XP_005548224. A referencepolypeptide sequence of cyno BTLA is disclosed in SEQ ID NO: 24. A cynoBTLA polypeptide sequence will typically have at least 90% sequenceidentity (such as at least 95%, 96%, 97%, 98%, 99% or 100%) to cyno BTLAas disclosed in SEQ ID NO: 24.

The term sequence identity is well known in the art. For the purposes ofthis invention, when determining whether a target sequence meets adefined limit (e.g. 90% identity), it is considered to meet the definedlimit if it is identified as such using the BLAST (Basic local alignmentsearch tool) algorithm (see Altschul et al. J Mol Biol 215:403-410,1990) or Smith-Waterman algorithm (see Smith and Waterman. J Mol. Biol.147:195-197, 1981).

Antibodies and Antigen-Binding Fragments of Antibodies

An antibody is an immunoglobulin molecule capable of specific binding toa target, such as a protein, polypeptide, peptide, carbohydrate,polynucleotide, lipid, or combinations of the foregoing through at leastone antigen recognition site, located in the variable domain of theimmunoglobulin molecule. In particular, as used herein, the term“antibody” encompasses intact polyclonal antibodies, intact monoclonalantibodies, multispecific antibodies such as bispecific antibodiesgenerated from at least two intact antibodies, chimeric antibodies,humanised antibodies, human antibodies, and any other modifiedimmunoglobulin molecule comprising an antigen recognition site so longas the antibodies exhibit the desired biological activity.

The term “antibody” as used herein, refers to an immunoglobulin moleculewhich specifically binds to an antigen and comprises an FcR binding sitewhich may or may not be functional.

As used herein, a BTLA agonist antibody (or antibody fragment) refers toan antibody (or antibody fragment) that binds to BTLA and enhances itscoinhibitory signal to T and/or B cells.

The antigen-binding site refers to the part of a molecule that binds toall or part of the target antigen. In an antibody molecule it may bereferred to as the antibody antigen-binding site and comprises the partof the antibody that specifically binds to all or part of the targetantigen.

Where an antigen is large, an antibody may only bind to a particularpart of the antigen, which part is termed an epitope. An antibodyantigen-binding site may be provided by one or more antibody variabledomains. Preferably, an antibody antigen-binding site comprises anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

The invention also encompassed antibody-fragments that comprise anantigen-binding site. Thus, the term “antigen-binding fragment thereof”,when referring to an antibody refers to antibody fragments, such as Fab,Fab′, F(ab′)2, diabodies, Fv fragments and single chain Fv (scFv)mutants that possess an antigen recognition site, and thus, the abilityto bind to an antigen. Antigen-binding immunoglobulin (antibody)fragments are well known in the art. Such fragment need not have afunctional Fc receptor binding site.

As used herein the terms “antibody fragment molecules of the invention”,“antibody fragment” and “antigen-binding fragment thereof”, are usedinterchangeably herein. The term “BTLA-binding molecule” as used herein,refers to both antibodies and binding fragments thereof capable ofbinding to BTLA.

There are five major classes (i.e., isotypes) of immunoglobulins: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided intosubclasses (subtypes), e.g., IgG1, lgG2, lgG3, lgG4, IgA1 and 1IgA2. Theheavy-chain constant regions that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.Unless dictated otherwise by contextual constraints the antibodies ofthe invention can be from one of these classes or subclasses ofantibodies. Heavy-chain constant domains that correspond to thedifferent classes of antibodies are typically denoted by thecorresponding lower-case Greek letter α, δ, ε, γ, and μ, respectively.Light chains of the antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino acid sequences of their constant domains.

“Native antibodies” are usually heterotetrameric Y-shaped glycoproteinsof about 150,000 daltons, composed of two identical light (L) chains andtwo identical heavy (H) chains. Each light chain is linked to a heavychain by one covalent disulfide bond, while the number of disulfidelinkages varies among the heavy chains of different immunoglobulinisotypes. Each heavy and light chain also has regularly spacedintrachain disulfide bridges. Each heavy chain has at one end a variabledomain (VH) followed by a number of constant domains. Each light chainhas a variable domain at one end (VL) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains. Each heavy chain comprises onevariable domain (VH) and a constant region, which in the case of IgG,IgA, and IgD antibodies, comprises three domains termed C_(H)1, C_(H)2,and C_(H)3 (IgM and IgE have a fourth domain, C_(H)4). In IgG, IgA, andIgD classes the C_(H)1 and C_(H)2 domains are separated by a flexiblehinge region, which is a proline and cysteine rich segment of variablelength (from about 10 to about 60 amino acids in various IgGsubclasses). The variable domains in both the light and heavy chains arejoined to the constant domains by a “J” region of about 12 or more aminoacids and the heavy chain also has a “D” region of about 10 additionalamino acids. Each class of antibody further comprises inter-chain andintra-chain disulfide bonds formed by paired cysteine residues. Theheavy chain variable region (VH) and light chain variable region (VL)can each be further subdivided into regions of hypervariability, termedCDRs, interspersed with regions that are more conserved, termedframework regions (FR). Each VH and VL, comprises three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g. effector cells) and the first component(Clq) of the classical complement system.

The antibody or antigen-binding fragment thereof of the invention may befrom any animal species including murine, rat, human, or any otherorigin (including chimeric or humanised antibodies). In someembodiments, the antibody or antigen-binding antibody fragment ismonoclonal, e,g. a monoclonal antibody. In some embodiments, theantibody or antigen-binding fragment thereof is a human or humanisedantibody or antigen-binding fragment thereof. A non-human antibody orantigen-binding fragment thereof may be humanised by recombinant methodsto reduce its immunogenicity in man.

An antibody or antigen-binding fragment thereof of the invention may beidentified using well-known methods. For example, the antigen-bindingmoiety may have been selected using phage display or otherantigen-binding selection or panning approach. Such antigen-bindingmoiety could then be incorporated into an antibody framework (e.g. fusedto the constant and hinge regions of, for example an IgG1 or IgG4molecule).

The term “monoclonal antibody” (“mAb”) as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, e.g., the individual antibodies comprising the populationare identical except for possible mutations, e.g., naturally occurringmutations, that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody or fragmentthereof, as not being a mixture of discrete antibodies orantigen-binding fragments. A mAb is highly specific, being directedagainst a single antigenic site/epitope.

A mAb may be produced by hybridoma, recombinant, transgenic or othertechniques known to those skilled in the art. For example, a monoclonalantibody or antigen-binding fragment thereof in accordance with thepresent invention may be made by the hybridoma method first described byKohler and Milstein (Nature 256:495, 1975) or may be made by recombinantDNA methods such as described in U.S. Pat. No. 4,816,567 and 6,331,415.The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature1991; 352:624-628 and Marks et al., J. Mol. Biol. 1991; 222:581-597, forexample.

The term monoclonal may also be ascribed to an antigen-binding fragmentof an antibody of the invention. It merely means that the molecule isproduced or present in a single clonal form.

A “human” antibody (HumAb) refers to an antibody having variable regionsin which both the framework and CDR regions are derived from humangermline immunoglobulin sequences. Furthermore, if the antibody containsa constant region, the constant region is also derived from humangermline immunoglobulin sequences. The human antibodies may includeamino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo). However, the term“human antibody,” as used herein, is not intended to include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

Human antibodies can be prepared by administering an immunogen/antigento a transgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge, but whose endogenous loci have been disabled,e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and6,150,584 regarding XENOMOUSE (trade mark) technology). See also, forexample, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006)regarding human antibodies generated via a human B-cell hybridomatechnology. Such animals typically contain all or a portion of the humanimmunoglobulin loci, which replace the endogenous immunoglobulin loci,or which are present extra chromosomally or integrated randomly into theanimal's chromosomes. In such transgenic mice, the endogenousimmunoglobulin loci have generally been inactivated. For review ofmethods for obtaining human antibodies from transgenic animals, seeLonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. PatentNos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Pat.No. 5,770,429 describing HUMAB™ technology; U.S. Pat. No. 7,041,870describing K-M MOUSE™ technology, and U.S. Patent ApplicationPublication No. US2007/0061900, describing VELOCIMOUSE™ technology.Human variable regions from intact antibodies generated by such animalsmay be further modified, e.g., by combining with a different humanconstant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol, 133:3001 (1984); Brodeur et al., Monoclonal Antibody ProductionTechniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York,1987); and Boerner et al., J. Immunol., 147:86 (1991)) Human antibodiesgenerated via human B-cell hybridoma technology are also described in Liet al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additionalmethods include those described, for example, in U.S. Pat. No. 7,189,826(describing production of monoclonal human IgM antibodies from hybridomacell lines) and Ni, Xiandai Mianyixue, 26:265-268 (2006) (describinghuman-human hybridomas). Human hybridoma technology (Trioma technology)is also described in Vollmers and Brandlein, Histology andHistopathology, 20:927-937 (2005) and Vollmers and Brandlein, Methodsand Findings in Experimental and Clinical Pharmacology, 27:185-91(2005).

The terms “human” antibodies and “fully human” antibodies are usedsynonymously. This definition of a human antibody specifically excludesa humanised antibody comprising non-human antigen-binding residues.

As used herein, a “humanised antibody” refers to an antibody in whichsome, most or all of the amino acids outside the CDRs of a non-humanantibody are replaced with corresponding amino acids derived from humanimmunoglobulins. In some embodiments, humanised antibodies are humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity. The humanised antibody may compriseresidues that are found neither in the recipient antibody nor in theimported CDR or framework sequences but are included to further refineand optimize antibody performance. In one embodiment of a humanised formof an Ab, some, most or all the amino acids outside the CDRs have beenreplaced with amino acids from human immunoglobulins, whereas some, mostor all amino acids within one or more CDR regions are unchanged. Smalladditions, deletions, insertions, substitutions or modifications ofamino acids are permissible provided they do not abrogate the ability ofthe antibody to bind to a particular antigen. A “humanised” antibodyretains an antigenic specificity similar to that of the originalantibody. In general, a humanised antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the hypervariable loops correspond to those of anon-human immunoglobulin, and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanised antibodyoptionally will also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see, e.g., Jones et al, Nature 321:522-525 (1986);Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr. Op.Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton,Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

As used herein, an “engineered antibody” refers to an antibody, whichmaybe a humanized antibody, wherein particular residues have beensubstituted for others so as to diminish an adverse effect or property.Such substitution could be within a CD domain. For example, as describedherein (see Example 21), the CDRH2 of the humanised antibody 3E8 wasmodified with an N57Q substitution to remove deamidation potential, anda K63S substitution to reduce predicted immunogenicity.

A “chimeric antibody” refers to an antibody in which the variableregions are derived from one species and the constant regions arederived from another species, such as an antibody in which the variableregions are derived from a mouse antibody and the constant regions arederived from a human antibody or vice versa. The term also encompassesan antibody comprising a V region from one individual from one species(e.g., a first mouse) and a constant region from another individual fromthe same species (e.g., a second mouse). The term “antigen (Ag)” refersto the molecular entity used for immunization of an immunocompetentvertebrate to produce the antibody (Ab) that recognizes the Ag or toscreen an expression library (e.g., phage, yeast or ribosome displaylibrary, among others). Herein, Ag is termed more broadly and isgenerally intended to include target molecules that are specificallyrecognized by the Ab, thus including portions or mimics of the moleculeused in an immunization process for raising the Ab or in libraryscreening for selecting the Ab.

A “bispecific” or “bifunctional” antibody is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Traditionally, the recombinant production of bispecificantibodies is based on the co-expression of two immunoglobulinheavy-chain/light-chain pairs, where the two heavy chains have differentspecificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Methodsfor making bispecific antibodies are within the purview of those skilledin the art. For example, bispecific antibodies can be produced by avariety of methods including fusion of hybridomas or linking of Fab′fragments. See, e.g., Songsivilai, et al, (1990) Clin. Exp. Immunol. 79:315-321, Kostelny, et al, (1992) J Immunol. 148:1547-1553. In addition,bispecific antibodies may be formed as “diabodies” (Holliger, et al,(1993) PNAS USA 90:6444-6448) or as “Janusins” (Traunecker, et al,(1991) EMBO J. 10:3655-3659 and Traunecker, et al, (1992) Int. J. CancerSuppl. 7:51-52).

Generally, the term “epitope” refers to the area or region of an antigento which an antibody specifically binds, i.e., an area or region inphysical contact with the antibody. Thus, the term “epitope” refers tothat portion of a molecule capable of being recognized by and bound byan antibody at one or more of the antibody's antigen-binding regions.Typically, an epitope is defined in the context of a molecularinteraction between an “antibody, or antigen-binding portion thereof”(Ab), and its corresponding antigen. Epitopes often consist of a surfacegrouping of molecules such as amino acids or sugar side chains and havespecific three-dimensional structural characteristics as well asspecific charge characteristics. In some embodiments, the epitope can bea protein epitope. Protein epitopes can be linear or conformational. Ina linear epitope, all of the points of interaction between the proteinand the interacting molecule (such as an antibody) occur linearly alongthe primary amino acid sequence of the protein. A “nonlinear epitope” or“conformational epitope” comprises non-contiguous polypeptides (or aminoacids) within the antigenic protein to which an antibody specific to theepitope binds. The term “antigenic epitope” as used herein, is definedas a portion of an antigen to which an antibody can specifically bind asdetermined by any method well known in the art, for example, byconventional immunoassays.

An antibody that “specifically binds” to an epitope is a term wellunderstood in the art, and methods to determine such specific bindingare also well known in the art. A molecule is said to exhibit “specificbinding” if it reacts or associates more frequently, more rapidly, withgreater duration and/or with greater affinity with a particular cell,protein or substance than it does with alternative cells, proteins orsubstances.

A variety of assay formats may be used to select an antibody or peptidethat specifically binds a molecule of interest. For example, solid-phaseELISA immunoassay, immunoprecipitation, Biacore™ (GE Healthcare,Piscataway, N.J.), KinExA, fluorescence-activated cell sorting (FACS),Octet™ (ForteBio, Inc., Menlo Park, Calif.) and Western blot analysisare among many assays that may be used to identify an antibody thatspecifically reacts with an antigen or a receptor, or ligand bindingportion thereof, that specifically binds with a cognate ligand orbinding partner. Typically, a specific or selective reaction will be atleast twice the background signal or noise, more typically more than 10times background, even more typically, more than 50 times background,more typically, more than 100 times background, yet more typically, morethan 500 times background, even more typically, more than 1000 timesbackground, and even more typically, more than 10,000 times background.Also, an antibody is said to “specifically bind” an antigen when theequilibrium dissociation constant (K_(D) or KD, as used interchangeablyherein) is <7 nM.

According to the first aspect of the invention there is provided anisolated antibody or an antigen-binding fragment thereof thatspecifically binds BTLA, wherein the antibody has a heavy chain and/orlight chain with at least one CDR an antibody selected from the groupconsisting of 11.5.1, 2.8.6, 12F11, 14D4, 15B6, 15C6, 16E1, 16F10, 16H2,1H6, 21C7, 24H7, 26B1, 26F3, 27G9, 3A9, 3E8, 4B1, 4D3, 4D5, 4E8, 4H4,6G8, 7A1, 8B4, 8C4, 6.2 and 831, as disclosed in Table 1 and describedherein. In one embodiment, the antibody or antigen-binding fragmentthereof competes for binding to BTLA with its natural ligand HVEM. Inanother embodiment, the antibody or antigen-binding fragment thereofdoes not interfere with binding of HVEM.

In additional embodiments, the isolated antibody which binds human BTLAis selected from the group consisting of 11.5.1 and 2.8.6, wherein theantibody specifically binds BTLA and induces signaling through thereceptor.

By an antibody selected from the group consisting of 11.5.1, 2.8.6,12F11, 14D4, 15B6, 15C6, 16E1, 16F10, 16H2, 1H6, 21C7, 24H7, 26B1, 26F3,27G9, 3A9, 3E8, 4B1, 4D3, 4D5, 4E8, 4H4, 6G8, 7A1, 8B4, 8C4, 6.2 and831, as disclosed in Table 1 and described herein, means any antibody orantigen-binding fragment thereof which comprises one or more, such as VHCDR 1, 2 and 3, or VL CDR 1, 2 and 3, or VH CDR 1, 2 and 3 and VL CDR 1,2 and 3, from any of the antibodies disclosed in Table 1 (whethermurine, humanized or humanised/engineered).

According to a variation of the first aspect of the invention there isprovided an isolated antibody or an antigen-binding fragment thereofthat specifically binds human BTLA comprising at least one VH CDR thathas an amino acid sequence as set forth in any of SEQ ID NO: 7, SEQ IDNO: 8 or SEQ ID NO: 9, with from 0 to 3 amino acid modifications, suchas 0, 1, 2, or 3 amino acid modifications. In certain embodiments, theamino acid modifications include, but not limited to, amino acidsubstitution, addition, deletion, or chemical modification, withouteliminating the antibody binding affinity or T-cell inhibitory effect ofthe modified amino acid sequence, as compared to the unmodified aminoacid sequence.

According to a variation of the first aspect of the invention there isprovided an isolated antibody or an antigen-binding fragment thereofthat specifically binds human BTLA comprising at least one VH CDR withan amino acid sequence as set forth in any of SEQ ID NO: 193, SEQ ID NO:194, or SEQ ID NO: 195, with from 0 to 3 amino acid modifications.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein the heavy chain comprises a heavy chain variableregion comprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1 hasan amino acid sequence as set forth in SEQ ID NO: 7, CDRH2 has an aminoacid sequence as set forth in SEQ ID NO: 8, and CDRH3 has an amino acidsequence as set forth in SEQ ID NO: 9.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein the heavy chain comprises a heavy chain variableregion comprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1 hasan amino acid sequence as set forth in SEQ ID NO: 193, CDRH2 has anamino acid sequence as set forth in SEQ ID NO: 194, and CDRH3 has anamino acid sequence as set forth in SEQ ID NO: 195.

According to a variation of the first aspect of the invention there isprovided an isolated antibody or an antigen-binding fragment thereofthat specifically binds human BTLA comprising at least one VL CDR withan amino acid sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 11 orSEQ ID NO: 12, with from 0 to 3 amino acid modifications.

According to a variation of the first aspect of the invention there isprovided an isolated antibody or an antigen-binding fragment thereofthat specifically binds human BTLA comprising at least one VL CDR withan amino acid sequence as set forth in SEQ ID NO: 196, SEQ ID NO: 197,or SEQ ID NO: 12, with from 0 to 3 amino acid modifications.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein the light chain comprises a light chain variableregion comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 hasan amino acid sequence as set forth in SEQ ID NO: 10, CDRL2 has an aminoacid sequence as set forth in SEQ ID NO: 11, and CDRL3 has an amino acidsequence as set forth in SEQ ID NO: 12.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein the light chain comprises a light chain variableregion comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 hasan amino acid sequence as set forth in SEQ ID NO: 196, CDRL2 has anamino acid sequence as set forth in SEQ ID NO: 197, and CDRL3 has anamino acid sequence as set forth in SEQ ID NO: 12.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein the heavy chain comprises a heavy chain variableregion comprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1 hasan amino acid sequence as set forth in SEQ ID NO: 7, CDRH2 has an aminoacid sequence as set forth in SEQ ID NO: 8, and CDRH3 has an amino acidsequence as set forth in SEQ ID NO: 9, and the light chain comprises alight chain variable region comprising three CDRs: CDRL1, CDRL2 andCDRL3, wherein CDRL1 has an amino acid sequence as set forth in SEQ IDNO: 10, CDRL2 has an amino acid sequence as set forth in SEQ ID NO: 11,and CDRL3 has an amino acid sequence as set forth in SEQ ID NO: 12.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein the heavy chain comprises a heavy chain variableregion comprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1 hasan amino acid sequence as set forth in SEQ ID NO: 193, CDRH2 has anamino acid sequence as set forth in SEQ ID NO: 194, and CDRH3 has anamino acid sequence as set forth in SEQ ID NO: 195, and the light chaincomprises a light chain variable region comprising three CDRs: CDRL1,CDRL2 and CDRL3, wherein CDRL1 has an amino acid sequence as set forthin SEQ ID NO: 196, CDRL2 has an amino acid sequence as set forth in SEQID NO: 197, and CDRL3 has an amino acid sequence as set forth in SEQ IDNO: 12.

According to a variation of the first aspect of the invention there isprovided an isolated antibody or an antigen-binding fragment thereofthat specifically binds human BTLA comprising at least one VH CDR withan amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQID NO: 3, with from 0 to 3 amino acid modifications.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA comprising at least one VHCDR with an amino acid sequence as set forth in SEQ ID NO: 199, SEQ IDNO: 200, or SEQ ID NO: 201, with from 0 to 3 amino acid modifications.

According to a variation of the first aspect of the invention there isprovided an isolated antibody or an antigen-binding fragment thereofthat specifically binds human BTLA, comprising a heavy chain and a lightchain, wherein the heavy chain comprises a heavy chain variable regioncomprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1 has anamino acid sequence as set forth in SEQ ID NO: 1, CDRH2 has an aminoacid sequence as set forth in SEQ ID NO: 2, and CDRH3 has an amino acidsequence as set forth in SEQ ID NO: 3.

According to a variation of the first aspect of the invention there isprovided an isolated antibody or an antigen-binding fragment thereofthat specifically binds human BTLA, comprising a heavy chain and a lightchain, wherein the heavy chain comprises a heavy chain variable regioncomprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1 has anamino acid sequence as set forth in SEQ ID NO: 199, CDRH2 has an aminoacid sequence as set forth in SEQ ID NO: 200, and CDRH3 has an aminoacid sequence as set forth in SEQ ID NO: 201.

According to a variation of the first aspect of the invention there isprovided an isolated antibody or an antigen-binding fragment thereofthat specifically binds human BTLA comprising at least one VL CDR withan amino acid sequence as set forth in SEQ ID NO: 4, SEQ ID NO: 5 or SEQID NO: 6.

According to a variation of the first aspect of the invention there isprovided an isolated antibody or an antigen-binding fragment thereofthat specifically binds human BTLA comprising at least one VL CDR withan amino acid sequence as set forth in SEQ ID NO: 202, SEQ ID NO: 203,or SEQ ID NO: 6.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein the light chain comprises a light chain variableregion comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 hasan amino acid sequence as set forth in SEQ ID NO: 4, CDRL2 has an aminoacid sequence as set forth in SEQ ID NO: 5, and CDRL3 has an amino acidsequence as set forth in SEQ ID NO: 6.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein the light chain comprises a light chain variableregion comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 hasan amino acid sequence as set forth in SEQ ID NO: 202, CDRL2 has anamino acid sequence as set forth in SEQ ID NO: 203, and CDRL3 has anamino acid sequence as set forth in SEQ ID NO: 6.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein the heavy chain comprises a heavy chain variableregion comprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1 hasan amino acid sequence as set forth in SEQ ID NO: 1, CDRH2 has an aminoacid sequence as set forth in SEQ ID NO: 2, and CDRH3 has an amino acidsequence as set forth in SEQ ID NO: 3, and the light chain comprises alight chain variable region comprising three CDRs: CDRL1, CDRL2 andCDRL3, wherein CDRL1 has an amino acid sequence as set forth in SEQ IDNO: 4, CDRL2 has an amino acid sequence as set forth in SEQ ID NO: 5,and CDRL3 has an amino acid sequence as set forth in SEQ ID NO: 6.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein the heavy chain comprises a heavy chain variableregion comprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1 hasan amino acid sequence as set forth in SEQ ID NO: 199, CDRH2 has anamino acid sequence as set forth in SEQ ID NO: 200, and CDRH3 has anamino acid sequence as set forth in SEQ ID NO: 201, and the light chaincomprises a light chain variable region comprising three CDRs: CDRL1,CDRL2 and CDRL3, wherein CDRL1 has an amino acid sequence as set forthin SEQ ID NO: 202, CDRL2 has an amino acid sequence as set forth in SEQID NO: 203, and CDRL3 has an amino acid sequence as set forth in SEQ IDNO: 6.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA comprising at least one VHCDR with an amino acid sequence as set forth in (1) SEQ ID NO: 31, 32,or 33, with from 0 to 3 amino acid modifications; (2) SEQ ID NO: 37, 38,or 39, with from 0 to 3 amino acid modifications; (3) SEQ ID NO: 43, 44,or 45, with from 0 to 3 amino acid modifications; (4) SEQ ID NO: 43, 56,or 57, with from 0 to 3 amino acid modifications; (5) SEQ ID NO: 61, 62,or 63, with from 0 to 3 amino acid modifications; (6) SEQ ID NO: 31, 32,or 69, with from 0 to 3 amino acid modifications; (7) SEQ ID NO: 73, 74,or 75, with from 0 to 3 amino acid modifications; (8) SEQ ID NO: 79, 80,or 81, with from 0 to 3 amino acid modifications; (9) SEQ ID NO: 85, 86,or 87, with from 0 to 3 amino acid modifications; (10) SEQ ID NO: 61,92, or 93, with from 0 to 3 amino acid modifications; (11) SEQ ID NO:97, 98, or 99, with from 0 to 3 amino acid modifications; (12) SEQ IDNO: 103, 104, or 105, with from 0 to 3 amino acid modifications; (13)SEQ ID NO: 109, 110, or 111, with from 0 to 3 amino acid modifications;(14) SEQ ID NO: 85, 110, or 117, with from 0 to 3 amino acidmodifications; (15) SEQ ID NO: 121, 122, or 123, with from 0 to 3 aminoacid modifications; (16) SEQ ID NO: 127, 128, or 129, with from 0 to 3amino acid modifications; (17) SEQ ID NO: 133, 134, or 135, with from 0to 3 amino acid modifications; (18) SEQ ID NO: 139, 140, or 141, withfrom 0 to 3 amino acid modifications; (19) SEQ ID NO: 145, 146, or 147,with from 0 to 3 amino acid modifications; (20) SEQ ID NO: 31, 32, or33, with from 0 to 3 amino acid modifications; (21) SEQ ID NO: 31, 32,or 159, with from 0 to 3 amino acid modifications; (22) SEQ ID NO: 169,170, or 171, with from 0 to 3 amino acid modifications; (23) SEQ ID NO:61, 62, or 63, with from 0 to 3 amino acid modifications; (24) SEQ IDNO: 31, 182, or 183, with from 0 to 3 amino acid modifications; (25) SEQID NO: 187, 188, or 189, with from 0 to 3 amino acid modifications; (26)SEQ ID NO: 193, 194, or 195, with from 0 to 3 amino acid modifications;(27) SEQ ID NO: 199, 200, or 201, with from 0 to 3 amino acidmodifications; (28) SEQ ID NO: 205, 206, or 207, with from 0 to 3 aminoacid modifications; (29) SEQ ID NO: 211, 212, or 213, with from 0 to 3amino acid modifications; (30) SEQ ID NO: 127, 386, or 129, with from 0to 3 amino acid modifications; (33) SEQ ID NO: 205, 206, or 207, withfrom 0 to 3 amino acid modifications; (34) SEQ ID NO: 127, 388, or 129,with from 0 to 3 amino acid modifications; or (35) SEQ ID NO: 205, 387,or 207, with from 0 to 3 amino acid modifications.

According to a variation of the first aspect of the invention there isprovided an isolated antibody or an antigen-binding fragment thereofthat specifically binds human BTLA, comprising a heavy chain and a lightchain, wherein the heavy chain comprises a heavy chain variable regioncomprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1, CDRH2,CDRH3 have an amino acid sequence as set forth in (1) SEQ ID NO: 31, SEQID NO: 32, and SEQ ID NO: 33, respectively; (2) SEQ ID NO: 37, SEQ IDNO: 38, and SEQ ID NO: 39, respectively; (3) SEQ ID NO: 43, SEQ ID NO:44, and SEQ ID NO: 45, respectively; (4) SEQ ID NO: 43, SEQ ID NO: 56,and SEQ ID NO: 57, respectively; (5) SEQ ID NO: 61, SEQ ID NO: 62, andSEQ ID NO: 63, respectively; (6) SEQ ID NO: 31, SEQ ID NO: 32, and SEQID NO: 69, respectively; (7) SEQ ID NO: 73, SEQ ID NO: 74, and SEQ IDNO: 75, respectively; (8) SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO:81, respectively; (9) SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87,respectively; (10) SEQ ID NO: 61, SEQ ID NO: 92, and SEQ ID NO: 93,respectively; (11) SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99,respectively; (12) SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105,respectively; (13) SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111,respectively; (14) SEQ ID NO: 85, SEQ ID NO: 110, and SEQ ID NO: 117,respectively; (15) SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123,respectively; (16) SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129,respectively; (17) SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135,respectively; (18) SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 141,respectively; (19) SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 147,respectively; (20) SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33,respectively; (21) SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 159,respectively; (22) SEQ ID NO: 169, SEQ ID NO: 170, and SEQ ID NO: 171,respectively; (23) SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63,respectively; (24) SEQ ID NO: 31, SEQ ID NO: 182, and SEQ ID NO: 183,respectively; (25) SEQ ID NO: 187, SEQ ID NO: 188, and SEQ ID NO: 189,respectively; (26) SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195,respectively; (27) SEQ ID NO: 199, SEQ ID NO: 200, and SEQ ID NO: 201,respectively; (28) SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207,respectively; (29) SEQ ID NO: 211, 212, and 213, respectively; (30) SEQID NO: 127, 386, and 129, respectively; (31) SEQ ID NO: 205, 206, and207, respectively; (32) SEQ ID NO: 127, 388, and 129, respectively; or(33) SEQ ID NO: 205, 387, and 207, respectively, wherein from 0 to 3amino acid modifications can be present in any CDR/SEQ ID NO:.

According to a variation of the first aspect of the invention there isprovided an isolated antibody or an antigen-binding fragment thereofthat specifically binds human BTLA comprising at least one VL CDR withan amino acid sequence as set forth in (1) SEQ ID NO: 34, 35, or 36; (2)SEQ ID NO: 40, 41, or 42; (3) SEQ ID NO: 46, 47, or 48; (4) SEQ ID NO:58, 59, or 60; (5) SEQ ID NO: 64, 65, or 66; (6) SEQ ID NO: 34, 35, or72; (7) SEQ ID NO: 76, 47, or 78; (8) SEQ ID NO: 82, 83, or 84; (9) SEQID NO: 88, 89, or 90; (10) SEQ ID NO: 94, 95, or 96; (11) SEQ ID NO:100, 101, or 102; (12) SEQ ID NO: 64, 107, or 108; (13) SEQ ID NO: 88,89, or 114; (14) SEQ ID NO: 124, 125, or 126; (15) SEQ ID NO: 34, 35, or36; (16) SEQ ID NO: 136, 137, or 138; (17) SEQ ID NO: 142, 143, or 144;(18) SEQ ID NO: 148, 149, or 150; ; (19) SEQ ID NO: 136, 137, or 162;(20) SEQ ID NO: 34, 35, or 36; (21) SEQ ID NO: 172, 173, or 174; (22)SEQ ID NO: 64, 65, or 180; (23) SEQ ID NO: 136, 137, or 186; (24) SEQ IDNO: 190, 191, or 192; (25) SEQ ID NO: 196, 197, or 12; (26) SEQ ID NO:202, 203, or 6; (27) SEQ ID NO: 142, 209, or 210; or (28) SEQ ID NO:214, 35, or 216; (29) SEQ ID NO: 10, 11, or 12; (30) SEQ ID NO: 4, 5, or6; or (31) SEQ ID NO: 142, 143, or 210, wherein from 0 to 3 amino acidmodifications can be present in any CDR/SEQ ID NO:.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein the light chain comprises a light chain variableregion comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1,CDRL2, and CDRL3 have an amino acid sequence as set forth in (1) SEQ IDNO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively; (2) SEQ ID NO:40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; (3) SEQ ID NO: 46,SEQ ID NO: 47, and SEQ ID NO: 48, respectively; (4) SEQ ID NO: 58, SEQID NO: 59, and SEQ ID NO: 60, respectively; (5) SEQ ID NO: 64, SEQ IDNO: 65, and SEQ ID NO: 66, respectively; (6) SEQ ID NO: 34, SEQ ID NO:35, and SEQ ID NO: 72, respectively; (7) SEQ ID NO: 76, SEQ ID NO: 47,and SEQ ID NO: 78, respectively; (8) SEQ ID NO: 82, SEQ ID NO: 83, andSEQ ID NO: 84, respectively; (9) SEQ ID NO: 88, SEQ ID NO: 89, and SEQID NO: 90, respectively; (10) SEQ ID NO: 94, SEQ ID NO: 95, and SEQ IDNO: 96, respectively; (11) SEQ ID NO: 100, SEQ ID NO: 101, and SEQ IDNO: 102, respectively; (12) SEQ ID NO: 64, SEQ ID NO: 107, and SEQ IDNO: 108, respectively; (13) SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO:114, respectively; (14) SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO:126, respectively; (15) SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36,respectively; (16) SEQ ID NO: 136, SEQ ID NO: 137, and SEQ ID NO: 138,respectively; (17) SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 144,respectively; (18) SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150,respectively; (19) SEQ ID NO: 136, SEQ ID NO: 137, and SEQ ID NO: 162,respectively; (20) SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36,respectively; (21) SEQ ID NO: 172, SEQ ID NO: 173, and SEQ ID NO: 174,respectively; (22) SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 180,respectively; (23) SEQ ID NO: 136, SEQ ID NO: 137, and SEQ ID NO: 186,respectively; (24) SEQ ID NO: 190, SEQ ID NO: 191, and SEQ ID NO: 192,respectively; (25) SEQ ID NO: 196, SEQ ID NO: 197, and SEQ ID NO: 12,respectively; (26) SEQ ID NO: 202, SEQ ID NO: 203, and SEQ ID NO: 6,respectively; (27) SEQ ID NO: 142, SEQ ID NO: 209, and SEQ ID NO: 210,respectively; (28) SEQ ID NO: 214, SEQ ID NO: 35, and SEQ ID NO: 216,respectively; (29) SEQ ID NO: 10, 11, or 12, respectively; (30) SEQ IDNO: 4, 5, or 6, respectively; or (31) SEQ ID NO: 142, 143, or 210,respectively, wherein from 0 to 3 amino acid modifications can bepresent in any CDR/SEQ ID NO:.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein the heavy chain comprises a heavy chain variableregion comprising three CDRs: CDRH1, CDRH2 and CDRH3, and the lightchain comprises a light chain variable region comprising three CDRs:CDRL1, CDRL2 and CDRL3, wherein (1) CDRH1, CDRH2, CDRH3 have an aminoacid sequence as set forth in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ IDNO: 33, respectively, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO:36, respectively; (2) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42,respectively; (3) CDRH1, CDRH2, CDRH3 have an amino acid sequence as setforth in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively,and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth inSEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively; (4)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 43, SEQ ID NO: 56, and SEQ ID NO: 57, respectively, and CDRL1,CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO:58, SEQ ID NO: 59, and SEQ ID NO: 60, respectively; (5) CDRH1, CDRH2,CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 61, SEQ IDNO: 62, and SEQ ID NO: 63, respectively, and CDRL1, CDRL2, and CDRL3have an amino acid sequence as set forth in SEQ ID NO: 64, SEQ ID NO:65, and SEQ ID NO: 66, respectively; (6) CDRH1, CDRH2, CDRH3 have anamino acid sequence as set forth in SEQ ID NO: 31, SEQ ID NO: 32, andSEQ ID NO: 69, respectively, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ IDNO: 72, respectively; (7) CDRH1, CDRH2, CDRH3 have an amino acidsequence as set forth in SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO:75, respectively, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 76, SEQ ID NO: 47, and SEQ ID NO:78, respectively; (8) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84,respectively; (9) CDRH1, CDRH2, CDRH3 have an amino acid sequence as setforth in SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively,and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth inSEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively; (10)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 61, SEQ ID NO: 92, and SEQ ID NO: 93, respectively, and CDRL1,CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO:94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively; (11) CDRH1, CDRH2,CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 97, SEQ IDNO: 98, and SEQ ID NO: 99, respectively, and CDRL1, CDRL2, and CDRL3have an amino acid sequence as set forth in SEQ ID NO: 100, SEQ ID NO:101, and SEQ ID NO: 102, respectively; (12) CDRH1, CDRH2, CDRH3 have anamino acid sequence as set forth in SEQ ID NO: 103, SEQ ID NO: 104, andSEQ ID NO: 105, respectively, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 64, SEQ ID NO: 107, and SEQ IDNO: 108, respectively; (13) CDRH1, CDRH2, CDRH3 have an amino acidsequence as set forth in SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO:111, respectively, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO:114, respectively; (14) CDRH1, CDRH2, CDRH3 have an amino acid sequenceas set forth in SEQ ID NO: 85, SEQ ID NO: 110, and SEQ ID NO: 117,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 114,respectively; (15) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 126,respectively; (16) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36,respectively; (17) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 136, SEQ ID NO: 137, and SEQ ID NO: 138,respectively; (18) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 141,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 144,respectively; (19) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 147,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150,respectively; (20) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150,respectively; (21) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 159,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 136, SEQ ID NO: 137, and SEQ ID NO: 162,respectively; (22) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 169, SEQ ID NO: 170, and SEQ ID NO: 171,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 172, SEQ ID NO: 173, and SEQ ID NO: 174,respectively; (23) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 180,respectively; (24) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 31, SEQ ID NO: 182, and SEQ ID NO: 183,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 136, SEQ ID NO: 137, and SEQ ID NO: 186,respectively; (25) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 187, SEQ ID NO: 188, and SEQ ID NO: 189,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 190, SEQ ID NO: 191, and SEQ ID NO: 192,respectively; (26) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 196, SEQ ID NO: 197, and SEQ ID NO: 12,respectively; (27) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 199, SEQ ID NO: 200, and SEQ ID NO: 201,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 202, SEQ ID NO: 203, and SEQ ID NO: 6,respectively; (28) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 142, SEQ ID NO: 209, and SEQ ID NO: 210,respectively; (29) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 211, SEQ ID NO: 212, and SEQ ID NO: 213,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 214, SEQ ID NO: 35, and SEQ ID NO: 216,respectively; (30) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 127, SEQ ID NO: 386, and SEQ ID NO: 129,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36,respectively; (31) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 142, SEQ ID NO: 209, and SEQ ID NO: 210,respectively; (32) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 127, SEQ ID NO: 388, and SEQ ID NO: 129,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36,respectively; (33) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 205, SEQ ID NO: 387, and SEQ ID NO: 207,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 142, SEQ ID NO: 209, and SEQ ID NO: 210,respectively; or (34) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 205, SEQ ID NO: 387, and SEQ ID NO: 207,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 210,respectively; wherein from 0 to 3 amino acid modifications can bepresent in any CDR/SEQ ID NO:.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising: (1) at least oneVH CDR with an amino acid sequence as set forth in SEQ ID NO: 31, SEQ IDNO: 32, or SEQ ID NO: 33, and at least one VL CDR with an amino acidsequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36;(2) at least one VH CDR with an amino acid sequence as set forth in SEQID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39, and at least one VL CDR withan amino acid sequence as set forth in SEQ ID NO: 40, SEQ ID NO: 41, orSEQ ID NO: 42; (3) at least one VH CDR with an amino acid sequence asset forth in SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45, and atleast one VL CDR with an amino acid sequence as set forth in SEQ ID NO:46, SEQ ID NO: 47, or SEQ ID NO: 48; (4) at least one VH CDR with anamino acid sequence as set forth in SEQ ID NO: 43, SEQ ID NO: 56, or SEQID NO: 57, and at least one VL CDR with an amino acid sequence as setforth in SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60; (5) at leastone VH CDR with an amino acid sequence as set forth in SEQ ID NO: 61,SEQ ID NO: 62, or SEQ ID NO: 63, and at least one VL CDR with an aminoacid sequence as set forth in SEQ ID NO: 64, SEQ ID NO: 65, or SEQ IDNO: 66; (6) at least one VH CDR with an amino acid sequence as set forthin SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 69, and at least one VLCDR with an amino acid sequence as set forth in SEQ ID NO: 34, SEQ IDNO: 35, or SEQ ID NO: 72; (7) at least one VH CDR with an amino acidsequence as set forth in SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 75,and at least one VL CDR with an amino acid sequence as set forth in SEQID NO: 76, SEQ ID NO: 47, or SEQ ID NO: 78; (8) at least one VH CDR withan amino acid sequence as set forth in SEQ ID NO: 79, SEQ ID NO: 80, orSEQ ID NO: 81, and at least one VL CDR with an amino acid sequence asset forth in SEQ ID NO: 82, SEQ ID NO: 83, or SEQ ID NO: 84; (9) atleast one VH CDR with an amino acid sequence as set forth in SEQ ID NO:85, SEQ ID NO: 86, or SEQ ID NO: 87, and at least one VL CDR with anamino acid sequence as set forth in SEQ ID NO: 88, SEQ ID NO: 89, or SEQID NO: 90; (10) at least one VH CDR with an amino acid sequence as setforth in SEQ ID NO: 61, SEQ ID NO: 92, or SEQ ID NO: 93, and at leastone VL CDR with an amino acid sequence as set forth in SEQ ID NO: 94,SEQ ID NO: 95, or SEQ ID NO: 96; (11) at least one VH CDR with an aminoacid sequence as set forth in SEQ ID NO: 97, SEQ ID NO: 98, or SEQ IDNO: 99, and at least one VL CDR with an amino acid sequence as set forthin SEQ ID NO: 100, SEQ ID NO: 101, or SEQ ID NO: 102; (12) at least oneVH CDR with an amino acid sequence as set forth in SEQ ID NO: 103, SEQID NO: 104, or SEQ ID NO: 105, and at least one VL CDR with an aminoacid sequence as set forth in SEQ ID NO: 64, SEQ ID NO: 107, or SEQ IDNO: 108; (13) at least one VH CDR with an amino acid sequence as setforth in SEQ ID NO: 109, SEQ ID NO: 110, or SEQ ID NO: 111, and at leastone VL CDR with an amino acid sequence as set forth in SEQ ID NO: 88,SEQ ID NO: 89, or SEQ ID NO: 114; (14) at least one VH CDR with an aminoacid sequence as set forth in SEQ ID NO: 85, SEQ ID NO: 110, or SEQ IDNO: 117, and at least one VL CDR with an amino acid sequence as setforth in SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 114; (15) at leastone VH CDR with an amino acid sequence as set forth in SEQ ID NO: 121,SEQ ID NO: 122, or SEQ ID NO: 123, and at least one VL CDR with an aminoacid sequence as set forth in SEQ ID NO: 124, SEQ ID NO: 125, or SEQ IDNO: 126; (16) at least one VH CDR with an amino acid sequence as setforth in SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 129, and at leastone VL CDR with an amino acid sequence as set forth in SEQ ID NO: 34,SEQ ID NO: 35, or SEQ ID NO: 36; (17) at least one VH CDR with an aminoacid sequence as set forth in SEQ ID NO: 133, SEQ ID NO: 134, or SEQ IDNO: 135, and at least one VL CDR with an amino acid sequence as setforth in SEQ ID NO: 136, SEQ ID NO: 137, or SEQ ID NO: 138; (18) atleast one VH CDR with an amino acid sequence as set forth in SEQ ID NO:139, SEQ ID NO: 140, or SEQ ID NO: 141, and at least one VL CDR with anamino acid sequence as set forth in SEQ ID NO: 142, SEQ ID NO: 143, orSEQ ID NO: 144; (19) at least one VH CDR with an amino acid sequence asset forth in SEQ ID NO: 145, SEQ ID NO: 146, or SEQ ID NO: 147, and atleast one VL CDR with an amino acid sequence as set forth in SEQ ID NO:148, SEQ ID NO: 149, or SEQ ID NO: 150; (20) at least one VH CDR with anamino acid sequence as set forth in SEQ ID NO: 31, SEQ ID NO: 32, or SEQID NO: 33, and at least one VL CDR with an amino acid sequence as setforth in SEQ ID NO: 148, SEQ ID NO: 149, or SEQ ID NO: 150; (21) atleast one VH CDR with an amino acid sequence as set forth in SEQ ID NO:31, SEQ ID NO: 32, or SEQ ID NO: 159, and at least one VL CDR with anamino acid sequence as set forth in SEQ ID NO: 136, SEQ ID NO: 137, orSEQ ID NO: 162; (22) at least one VH CDR with an amino acid sequence asset forth in SEQ ID NO: 169, SEQ ID NO: 170, or SEQ ID NO: 171, and atleast one VL CDR with an amino acid sequence as set forth in SEQ ID NO:172, SEQ ID NO: 173, or SEQ ID NO: 174; (23) at least one VH CDR with anamino acid sequence as set forth in SEQ ID NO: 61, SEQ ID NO: 62, or SEQID NO: 63, and at least one VL CDR with an amino acid sequence as setforth in SEQ ID NO: 64, SEQ ID NO: 65, or SEQ ID NO: 180; (24) at leastone VH CDR with an amino acid sequence as set forth in SEQ ID NO: 31,SEQ ID NO: 182, or SEQ ID NO: 183, and at least one VL CDR with an aminoacid sequence as set forth in SEQ ID NO: 136, SEQ ID NO: 137, or SEQ IDNO: 186; (25) at least one VH CDR with an amino acid sequence as setforth in SEQ ID NO: 187, SEQ ID NO: 188, or SEQ ID NO: 189, and at leastone VL CDR with an amino acid sequence as set forth in SEQ ID NO: 190,SEQ ID NO: 191, or SEQ ID NO: 192; (26) at least one VH CDR with anamino acid sequence as set forth in SEQ ID NO: 193, SEQ ID NO: 194, orSEQ ID NO: 195, and at least one VL CDR with an amino acid sequence asset forth in SEQ ID NO: 196, SEQ ID NO: 197, or SEQ ID NO: 12; (27) atleast one VH CDR with an amino acid sequence as set forth in SEQ ID NO:199, SEQ ID NO: 200, or SEQ ID NO: 201, and at least one VL CDR with anamino acid sequence as set forth in SEQ ID NO: 202, SEQ ID NO: 203, orSEQ ID NO: 6; (28) at least one VH CDR with an amino acid sequence asset forth in SEQ ID NO: 205, SEQ ID NO: 206, or SEQ ID NO: 207, and atleast one VL CDR with an amino acid sequence as set forth in SEQ ID NO:142, SEQ ID NO: 209, or SEQ ID NO: 210; (29) at least one VH CDR with anamino acid sequence as set forth in SEQ ID NO: 211, SEQ ID NO: 212, orSEQ ID NO: 213, and at least one VL CDR with an amino acid sequence asset forth in SEQ ID NO: 214, SEQ ID NO: 35, or SEQ ID NO: 216; (30) atleast one VH CDR with an amino acid sequence as set forth in SEQ ID NO:127, SEQ ID NO: 386, or SEQ ID NO: 129, and at least one VL CDR with anamino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35, or SEQID NO: 36; (31) at least one VH CDR with an amino acid sequence as setforth in SEQ ID NO: 205, SEQ ID NO: 206, or SEQ ID NO: 207, and at leastone VL CDR with an amino acid sequence as set forth in SEQ ID NO:142,SEQ ID NO: 209, or SEQ ID NO:210; (32) at least one VH CDR with an aminoacid sequence as set forth in SEQ ID NO: 127, SEQ ID NO: 388, or SEQ IDNO: 129, and at least one VL CDR with an amino acid sequence as setforth in SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36; (33) at leastone VH CDR with an amino acid sequence as set forth in SEQ ID NO: 205,SEQ ID NO: 387, or SEQ ID NO: 207, and at least one VL CDR with an aminoacid sequence as set forth in SEQ ID NO:142, SEQ ID NO: 209, or SEQ IDNO:210; or (34) at least one VH CDR with an amino acid sequence as setforth in SEQ ID NO: 205, SEQ ID NO: 387, or SEQ ID NO: 207, and at leastone VL CDR with an amino acid sequence as set forth in SEQ ID NO:142,SEQ ID NO: 143, or SEQ ID NO:210; wherein from 0 to 3 amino acidmodifications can be present in any CDR/SEQ ID NO:.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence as set forth in SEQ ID NO: 13,or a sequence with at least 90% sequence identity thereto. In otherembodiments, the heavy chain variable region comprises an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 17 or 21.

For any embodiment disclosed herein that refers to at least 90% sequenceidentity it is understood that this includes any sequence identity from90% to 100%, i.e. at least 90% at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% and 100%.

In other embodiments, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 301, 302, 303, 305, 306, 307, 308,309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,324, 306, 326, 327, 330, 331, 13, 17, 21, 382, 384, 389, 390 and 378.

In other embodiments, the heavy chain variable region comprises an aminoacid sequence as set forth in SEQ ID NO: 301, 302, 303, 305, 306, 307,308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,322, 324, 306, 326, 327, 330, 331, 13, 17, 21, 382, 384, 389, 390 and378, with up to 10 modifications, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 amino acid modifications therein.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein the light chain comprises a light chain variableregion comprising an amino acid sequence as set forth in SEQ ID NO: 14,or a sequence with at least 90% sequence identity thereto. In otherembodiments, the light chain variable region comprises an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 15 or 19.

In other embodiments, the light chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 351, 352, 353, 355, 356, 357, 358,359, 360, 361, 362, 363, 364, 366, 367, 368, 369, 370, 372, 351, 374,375, 376, 377, 380,381, 14, 15, 19, 383, 385 or 378.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence as set forth in SEQ ID NO: 13,and the light chain comprises a light chain variable region comprisingan amino acid sequence as set forth in SEQ ID NO: 14.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 13, andthe light chain comprises a light chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 14.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein: (1) the heavy chain comprises a heavy chainvariable region comprising an amino acid sequence that has at least 90%sequence identity to the amino acid sequence as set forth in SEQ ID NO:17, and the light chain comprises a light chain variable regioncomprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 15; or(2) the heavy chain comprises a heavy chain variable region comprisingan amino acid sequence that has at least 90% sequence identity to theamino acid sequence as set forth in SEQ ID NO: 21, and the light chaincomprises a light chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 19.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof that specifically binds human BTLA, comprising a heavy chain anda light chain, wherein: (1) the heavy chain comprises a heavy chainvariable region comprising an amino acid sequence that has at least 90%sequence identity to the amino acid sequence as set forth in SEQ ID NO:301, and the light chain comprises a light chain variable regioncomprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 351; (2)the heavy chain comprises a heavy chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 302, and the light chaincomprises a light chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 352; (3) the heavy chain comprises aheavy chain variable region comprising an amino acid sequence as setforth in SEQ ID NO: 303, and the light chain comprises a light chainvariable region comprising an amino acid sequence that has at least 90%sequence identity to the amino acid sequence as set forth in SEQ ID NO:353; (4) the heavy chain comprises a heavy chain variable regioncomprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 305, andthe light chain comprises a light chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 355; (5) the heavy chaincomprises a heavy chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 306, and the light chain comprises alight chain variable region comprising an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence as set forthin SEQ ID NO: 356; (6) the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 307, andthe light chain comprises a light chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 357; (7) the heavy chaincomprises a heavy chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 308, and the light chain comprises alight chain variable region comprising an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence as set forthin SEQ ID NO: 358; (8) the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 309, andthe light chain comprises a light chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 359; (9) the heavy chaincomprises a heavy chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 310, and the light chain comprises alight chain variable region comprising an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence as set forthin SEQ ID NO: 360; (10) the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 311, andthe light chain comprises a light chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 361; (11) the heavy chaincomprises a heavy chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 312, and the light chain comprises alight chain variable region comprising an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence as set forthin SEQ ID NO: 362; (12) the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 313, andthe light chain comprises a light chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 363; (13) the heavy chaincomprises a heavy chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 314, and the light chain comprises alight chain variable region comprising an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence as set forthin SEQ ID NO: 364; (14) the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 315, andthe light chain comprises a light chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 364; (15) the heavy chaincomprises a heavy chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 316, and the light chain comprises alight chain variable region comprising an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence as set forthin SEQ ID NO: 366; (16) the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 317, andthe light chain comprises a light chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 367; (17) the heavy chaincomprises a heavy chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 318, and the light chain comprises alight chain variable region comprising an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence as set forthin SEQ ID NO: 368; (18) the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 319, andthe light chain comprises a light chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 369; (19) the heavy chaincomprises a heavy chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 320, and the light chain comprises alight chain variable region comprising an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence as set forthin SEQ ID NO: 370; (20) the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 321, andthe light chain comprises a light chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 370; (21) the heavy chaincomprises a heavy chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 322, and the light chain comprises alight chain variable region comprising an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence as set forthin SEQ ID NO: 372; (22) the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 324, andthe light chain comprises a light chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 374; (23) the heavy chaincomprises a heavy chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 306, and the light chain comprises alight chain variable region comprising an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence as set forthin SEQ ID NO: 375; (24) the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 326, andthe light chain comprises a light chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 376; (25) the heavy chaincomprises a heavy chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 327, and the light chain comprises alight chain variable region comprising an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence as set forthin SEQ ID NO: 377; (26) the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 17, andthe light chain comprises a light chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 15; (27) the heavy chaincomprises a heavy chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 21, and the light chain comprises alight chain variable region comprising an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence as set forthin SEQ ID NO: 19; (28) the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 330, andthe light chain comprises a light chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 380; (29) the heavy chaincomprises a heavy chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 331, and the light chain comprises alight chain variable region comprising an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence as set forthin SEQ ID NO: 381; (30) the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 382, andthe light chain comprises a light chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 383; (31) the heavy chaincomprises a heavy chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 384, and the light chain comprises alight chain variable region comprising an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence as set forthin SEQ ID NO: 385; (32) the heavy chain comprises a heavy chain variableregion comprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 389, andthe light chain comprises a light chain variable region comprising anamino acid sequence that has at least 90% sequence identity to the aminoacid sequence as set forth in SEQ ID NO: 383; (33) the heavy chaincomprises a heavy chain variable region comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 390, and the light chain comprises alight chain variable region comprising an amino acid sequence that hasat least 90% sequence identity to the amino acid sequence as set forthin SEQ ID NO: 385; or (34) the heavy chain comprises a heavy chainvariable region comprising an amino acid sequence that has at least 90%sequence identity to the amino acid sequence as set forth in SEQ ID NO:390, and the light chain comprises a light chain variable regioncomprising an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 378.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 13 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 14.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 17 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 15.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 21 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 19.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 301 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 351.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 302 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 352.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 303 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 353.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 305 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 355.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 306 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 356.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 307 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 357.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 308 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 358.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 309 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 359.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 310 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 360.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 311 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 361.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 312 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 362.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 313 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 363.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 314 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 364.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 315 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 364.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 316 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 366.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 317 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 367.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 318 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 368.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 319 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 369.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 320 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 370.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 321 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 370.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 322 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 372.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 324 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 374.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 306 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 375.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 326 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 376.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 327 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 377.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 17 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 15.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 21 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 19.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 330 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 380.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 331 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 381.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 382 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 383.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 384 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 385.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 389 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 383.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 390 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 385.

In one embodiment, the heavy chain variable region comprises an aminoacid sequence that has at least 90% sequence identity to the amino acidsequence as set forth in SEQ ID NO: 390 and the light chain variableregion comprises an amino acid sequence that has at least 90% sequenceidentity to the amino acid sequence as set forth in SEQ ID NO: 378.

In other embodiments, the heavy chain variable region polypeptide has atleast 92%, at least 95%, at least 97%, at least 98% or at least 99%identity with the sequence disclosed in SEQ ID NO: 13.

In other embodiments, the heavy chain variable region polypeptide has atleast 92%, at least 95%, at least 97%, at least 98% or at least 99%identity with the sequence disclosed in SEQ ID NO: 17.

In other embodiments, the heavy chain variable region polypeptide has atleast 92%, at least 95%, at least 97%, at least 98% or at least 99%identity with the sequence disclosed in SEQ ID NO: 21.

In other embodiments, the heavy chain variable region polypeptide has atleast 92%, at least 95%, at least 97%, at least 98% or at least 99%identity with the sequence disclosed in SEQ ID NO: 301, 302, 303, 305,306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319,320, 321, 322, 324, 306, 326, 327, 330, 331, 382, 384, 389, or 390.

In other embodiments, the light chain variable region polypeptide has atleast 92%, at least 95%, at least 97%, at least 98% or at least 99%identity with the sequence disclosed in SEQ ID NO: 14.

In other embodiments, the light chain variable region polypeptide has atleast 92%, at least 95%, at least 97%, at least 98% or at least 99%identity with the sequence disclosed in SEQ ID NO: 15.

In other embodiments, the light chain variable region polypeptide has atleast 92%, at least 95%, at least 97%, at least 98% or at least 99%identity with the sequence disclosed in SEQ ID NO: 19.

In other embodiments, the light chain variable region polypeptide has atleast 92%, at least 95%, at least 97%, at least 98% or at least 99%identity with the sequence disclosed in SEQ ID NO: 351, 352, 353, 355,356, 357, 358, 359, 360, 361, 362, 363, 364, 364, 366, 367, 368, 369,370, 372, 374, 375, 376, 377, 380, 381, 383, 385, or 378.

According to another variation of the first aspect of the inventionthere is provided an isolated antibody or an antigen-binding fragmentthereof having primary VH domain, primary VL domain, CDRH1, CDRH2,CDRH3, CDRL1, CDRL2, and CDRL3 of any antibody clone as set forth inTable 1. In certain embodiments, provided herein is an isolated antibodyselected from the antibody clones as set forth in Table 1.

TABLE 1 Exemplary BTLA Agonistic Antibodies SEQ ID NOs CDR CDR CDR CDRCDR CDR Clone Scheme H1 H2 H3 L1 L2 L3 VH VL 10B1 Kabat 31 32 33 34 3536 301 351 12F11 Kabat 37 38 39 40 41 42 302 352 14D4 Kabat 43 44 45 4647 48 303 353 15B6 Kabat 43 56 57 58 59 60 305 355 15C6 Kabat 61 62 6364 65 66 306 356 16E1 Kabat 31 32 69 34 35 72 307 357 16F10 Kabat 73 7475 76 47 78 308 358 16H2 Kabat 79 80 81 82 83 84 309 359 1H6 Kabat 85 8687 88 89 90 310 360 21C7 Kabat 61 92 93 94 95 96 311 361 24H7 Kabat 9798 99 100 101 102 312 362 26B1 Kabat 103 104 105 64 107 108 313 363 26F3Kabat 109 110 111 88 89 114 314 364 27G9 Kabat 85 110 117 88 89 114 315364 3A9 Kabat 121 122 123 124 125 126 316 366 3E8 Kabat 127 128 129 3435 36 317 367 4B1 Kabat 133 134 135 136 137 138 318 368 4D3 Kabat 139140 141 142 143 144 319 369 4D5 Kabat 145 146 147 148 149 150 320 3704E8 Kabat 31 32 33 148 149 150 321 370 4H4 Kabat 31 32 159 136 137 162322 372 6G8 Kabat 169 170 171 172 173 174 324 374 7A1 Kabat 61 62 63 6465 180 306 375 8B4 Kabat 31 182 183 136 137 186 326 376 8C4 Kabat 187188 189 190 191 192 327 377 2.8.6 Kabat 193 194 195 196 197 12 17 1511.5.1 Kabat 199 200 201 202 203 6 21 19 6.2 Kabat 205 206 207 142 209210 330 380 831 Kabat 211 212 213 214 35 216 331 381 humanised 2.8.6IMGT 7 8 9 10 11 12 13 14 2.8.6 IMGT 7 8 9 10 11 12 17 15 11.5.1 IMGT 12 3 4 5 6 21 19 Humanised 3E8 Kabat 127 386 129 34 35 36 382 383Humanised 6.2 Kabat 205 206 207 142 209 210 384 385 Engineered Kabat 127388 129 34 35 36 389 383 humanised 3E8 Engineered humanised Kabat 205387 207 142 209 210 390 385 6.2 (2^(nd)) Engineered Kabat 205 387 207142 143 210 390 378 humanised 6.2

In particular embodiments, the heavy chain or light chain also comprisea constant region. If the molecule is a full-length IgG-type antibodymolecule, the heavy chain may comprise three constant domains.

In certain embodiments the isolated antibody or an antigen-bindingfragment thereof that specifically binds human BTLA exhibits a K_(D) forbinding to human BTLA of at most about 10×10⁻⁹M. In certain embodimentsthe isolated antibody or an antigen-binding fragment thereof thatspecifically binds human BTLA exhibits a K_(D) for binding to human BTLAof at most about 4×10⁻⁹M. In certain embodiments the isolated antibodyor an antigen-binding fragment thereof that specifically binds humanBTLA exhibits a K_(D) for binding to human BTLA of at most about1×10⁻⁹M.

In certain embodiments, an isolated antibody (e.g., humanised) of theinvention binds human BTLA at 37° C. with a K_(D) of no more than about10 nM (1×10⁻⁸M); suitably no more than about 1 nM; more suitably areembodiments in which the antibodies have K_(D) values at 37° C. of nomore than about 500 pM (5×10⁻¹⁰ M), 200 pM, 100 pM, 50 pM, 20 pM, 10 pM,5 pM or even 2 pM. The term “about”, as used in this context means+/−10%.

In certain embodiments, an isolated antibody (e.g., humanised) of theinvention binds human BTLA at 37° C. with an on rate of at least 1.0×10⁵(1/Ms). In certain embodiments, an isolated antibody (e.g., humanised)of the invention binds human BTLA at 37° C. with an on rate of at least2.0×10⁵ (1/Ms), 3.0×10⁵ (1/Ms), 4.0×10⁵ (1/Ms), 5.0×10⁵ (1/Ms), 6.0×10⁵(1/Ms), or 7.0×10⁵(1/Ms).

In certain embodiments, an isolated antibody (e.g., humanised) of theinvention binds human BTLA at 37° C. with an off rate of no more than orless than 1.0×10⁻³ (1/s). In certain embodiments, an isolated antibody(e.g., humanised) of the invention binds human BTLA at 37° C. with anoff rate of no more than or less than 3.0×10⁻⁴ (1/s). In certainembodiments, an isolated antibody (e.g., humanised) of the inventionbinds human BTLA at 37° C. with an off rate of no more than or less than2.0×10⁻⁴ (1/s), or 1.0×10⁻⁴ (1/s).

In particular embodiments of the first aspect of the invention, providedherein are isolated agonistic antibodies or antigen binding fragmentsthereof that specifically binds human B and T Lymphocyte Attenuator(BTLA) with a KD of less than 10 nM, as determined by surface plasmonresonance (SPR) at 37° C. using a method such as that described inExample 2, and wherein said antibody binds cynomolgus BTLA with a KD ofless than 20 nM, as determined by surface plasmon resonance (SPR) at 37°C. using a method such as that described in Example 2; does not inhibitbinding of BTLA to herpes virus entry mediator (HVEM), as determined forexample by surface plasmon resonance (SPR) using a method such as thatdescribed in Example 4; and inhibits proliferation of T cells in vitro,as determined for example by a mixed lymphocyte reaction assay using amethod such as that described in Example 9. In some embodiments, saidantibody or antigen binding fragment thereof binds human B and TLymphocyte Attenuator (BTLA) with an on rate of at least 5.0×10⁵ (1/Ms)as determined by surface plasmon resonance (SPR) at 37° C. using amethod such as that described in Example 2. In some embodiments, saidantibody or antigen binding fragment thereof binds human B and TLymphocyte Attenuator (BTLA) with an off rate of less than 3.0×10⁻⁴(1/s) as determined by surface plasmon resonance (SPR) at 37° C. using amethod such as that described in Example 2. In some embodiments, saidantibody or antigen binding fragment thereof binds human B and TLymphocyte Attenuator (BTLA) with an off rate from 3.0×10⁻⁴ (1/s) to1.0×10⁻³ (1/s) as determined by surface plasmon resonance (SPR) at 37°C. using a method such as that described in Example 2. In someembodiments, the antibody or antigen-binding fragment thereof binds aresidue of human BTLA selected from: D52, P53, E55, E57, E83, Q86, E103,L106 and E92 as determined by x-ray crystallography or by flow cytometryof mutated receptors using a method such as that described in Example 5.In some embodiments, the antibody or antigen-binding fragment thereofbinds a residue of human BTLA selected from: Y39, K41, R42, Q43, E45 andS47. In some embodiments, the antibody or antigen-binding fragmentthereof binds a residue of human BTLA selected from: D35, T78, K81, S121and L123. In some embodiments, the antibody or antigen-binding fragmentthereof binds residue H68 of human BTLA. In some embodiments, theantibody or antigen-binding fragment thereof binds a residue of humanBTLA selected from: N65 and A64.

Methods for characterizing the properties of an antibody orantigen-binding fragment thereof of the invention are well known in theart. A suitable method for determining binding specificity using surfaceplasmon resonance (SPR) at 37° C. is described in Example 2. A suitablemethod for determining whether the tested antibody/fragment thereofinhibits binding of BTLA to herpes virus entry mediator (HVEM) isdescribed in Example 4; this also employs surface plasmon resonance(SPR). A suitable method for determining whether the testedantibody/fragment thereof inhibits proliferation of T cells in vitro, isa mixed lymphocyte reaction assay such as that described in Example 9.Suitable methods for determining the site of binding of anantibody/fragment thereof to BTLA can utilise x-ray crystallography orflow cytometry of mutated receptors, such as by the method described inExample 5

In particular embodiments of the first aspect of the invention, providedherein are isolated agonistic antibodies or antigen binding fragmentsthereof that specifically binds human B and T Lymphocyte Attenuator(BTLA) with an on rate of at least 5.0×10⁵ (1/Ms), as determined bysurface plasmon resonance (SPR) at 37° C. using a method such as thatdescribed in Example 2, wherein said antibody does not inhibit bindingof BTLA to herpes virus entry mediator (HVEM) as determined for exampleby surface plasmon resonance (SPR) using a method such as that describedin Example 4; and wherein said antibody inhibits proliferation of Tcells in vitro, as determined for example by a mixed lymphocyte reactionassay using a method such as that described in Example 9. In someembodiments, said antibody or antigen binding fragment thereof bindshuman B and T Lymphocyte Attenuator (BTLA) with an off rate of less than3.0×10⁻⁴ (1/s) as determined by surface plasmon resonance (SPR) at 37°C. using a method such as that described in Example 2. In someembodiments, said antibody or antigen binding fragment thereof bindshuman B and T Lymphocyte Attenuator (BTLA) with a KD of less than 10 nM,as determined by surface plasmon resonance (SPR) at 37° C. using amethod such as that described in Example 2. In some embodiments, saidantibody or antigen binding fragment thereof binds cynomolgus BTLA witha KD of less than 20 nM, as determined by surface plasmon resonance(SPR) at 37° C. using a method such as that described in Example 2. Insome embodiments, the antibody or antigen-binding fragment thereof bindsa residue of human BTLA selected from: D52, P53, E55, E57, E83, Q86,E103, L106 and E92 as determined by x-ray crystallography or by flowcytometry of mutated receptors using a method such as that described inExample 5. In some embodiments, the antibody or antigen-binding fragmentthereof binds a residue of human BTLA selected from: Y39, K41, R42, Q43,E45 and S47. In some embodiments, the antibody or antigen-bindingfragment thereof binds a residue of human BTLA selected from: D35, T78,K81, S121 and L123. In some embodiments, the antibody or antigen-bindingfragment thereof binds residue H68 of human BTLA. In some embodiments,the antibody or antigen-binding fragment thereof binds a residue of BTLAselected from: N65 and A64.

In particular embodiments of the first aspect of the invention, providedherein are isolated agonistic antibody or antigen binding fragmentthereof that specifically binds human B and T Lymphocyte Attenuator(BTLA) with an off rate from 3.0×10⁻⁴ (1/s) to 1.0×10⁻³ (1/s) asdetermined by surface plasmon resonance (SPR) at 37° C. using a methodsuch as that described in Example 2, wherein said antibody does notinhibit binding of BTLA to herpes virus entry mediator (HVEM) asdetermined for example by surface plasmon resonance (SPR) using a methodsuch as that described in Example 4; and wherein said antibody inhibitsproliferation of T cells in vitro, as determined for example by a mixedlymphocyte reaction assay using a method such as that described inExample 9. In some embodiments, said antibody or antigen bindingfragment thereof binds human B and T Lymphocyte Attenuator (BTLA) with aKD of less than 10 nM, as determined by surface plasmon resonance (SPR)at 37° C. using a method such as that described in Example 2. In someembodiments, said antibody or antigen binding fragment thereof bindscynomolgus BTLA with a KD of less than 20 nM, as determined by surfaceplasmon resonance (SPR) at 37° C. using a method such as that describedin Example 2. In some embodiments, said antibody or antigen bindingfragment thereof binds human B and T Lymphocyte Attenuator (BTLA) withan on rate of at least 5.0×10⁵ (1/Ms) as determined by surface plasmonresonance (SPR) at 37° C. using a method such as that described inExample 2. In some embodiments, the antibody or antigen-binding fragmentthereof binds a residue of human BTLA selected from: D52, P53, E55, E57,E83, Q86, E103, L106 and E92 as determined by x-ray crystallography orby flow cytometry of mutated receptors using a method such as thatdescribed in Example 5. In some embodiments, the antibody orantigen-binding fragment thereof binds a residue of human BTLA selectedfrom: Y39, K41, R42, Q43, E45 and S47. In some embodiments, the antibodyor antigen-binding fragment thereof binds a residue of human BTLAselected from: D35, T78, K81, S121 and L123. In some embodiments, theantibody or antigen-binding fragment thereof binds residue H68 of humanBTLA. In some embodiments, the antibody or antigen-binding fragmentthereof binds a residue of human BTLA selected from: N65 and A64.

In particular embodiments of the first aspect of the invention, providedherein are isolated agonistic antibodies or antigen binding fragmentsthereof that specifically binds human B and T Lymphocyte Attenuator(BTLA) with an off rate of less than 1.0×10⁻³ (1/s) and an on rate of atleast 5.0×10⁵ (1/Ms), each as measured by surface plasmon resonance(SPR) at 37° C. using a method such as that described in Example 2,wherein said antibody does not inhibit binding of BTLA to herpes virusentry mediator (HVEM) as determined for example by surface plasmonresonance (SPR) using a method such as that described in Example 4; andwherein said antibody inhibits proliferation of T cells in vitro, asdetermined for example by a mixed lymphocyte reaction assay using amethod such as that described in Example 9. In some embodiments, saidantibody or antigen binding fragment thereof binds human B and TLymphocyte Attenuator (BTLA) with a KD of less than 10 nM, as determinedby surface plasmon resonance (SPR) at 37° C. using a method such as thatdescribed in Example 2. In some embodiments, said antibody or antigenbinding fragment thereof binds cynomolgus BTLA with a KD of less than 20nM, as determined by surface plasmon resonance (SPR) at 37° C. using amethod such as that described in Example 2. In some embodiments, theantibody or antigen-binding fragment thereof binds a residue of humanBTLA selected from: D52, P53, E55, E57, E83, Q86, E103, L106 and E92 asdetermined by x-ray crystallography or by flow cytometry of mutatedreceptors using a method such as that described in Example 5. In someembodiments, the antibody or antigen-binding fragment thereof binds aresidue of BTLA selected from: Y39, K41, R42, Q43, E45 and S47. In someembodiments, the antibody or antigen-binding fragment thereof binds aresidue of human BTLA selected from: D35, T78, K81, S121 and L123. Insome embodiments, the antibody or antigen-binding fragment thereof bindsresidue H68 of human BTLA. In some embodiments, the antibody orantigen-binding fragment thereof binds a residue of human BTLA selectedfrom: N65 and A64.

In particular embodiments of the first aspect of the invention, providedherein are isolated agonistic antibodies or antigen binding fragmentsthereof that specifically binds human B and T Lymphocyte Attenuator(BTLA) with a KD of less than 2 nM, as determined by surface plasmonresonance (SPR) at 37° C. using a method such as that described inExample 2, wherein said antibody inhibits binding of BTLA to herpesvirus entry mediator (HVEM) as determined by surface plasmon resonance(SPR) using a method such as that described in Example 4; and inhibitsproliferation of T cells in vitro, as determined for example by a mixedlymphocyte reaction assay using a method such as that described inExample 9. In some embodiments, said antibody or antigen bindingfragment thereof binds human B and T Lymphocyte Attenuator (BTLA) withan on rate of less than 1.0×10⁶ (1/Ms), as determined by surface plasmonresonance (SPR) at 37° C. using a method such as that described inExample 2. In some embodiments, said antibody or antigen bindingfragment thereof binds human B and T Lymphocyte Attenuator (BTLA) withan off rate of less than 1.0×10⁻³ (1/s), as determined by surfaceplasmon resonance (SPR) at 37° C. using a method such as that describedin Example 2. In some embodiments, said antibody or antigen bindingfragment thereof binds cynomolgus B and T Lymphocyte Attenuator (BTLA)with a KD of less than 10 nM, as determined by surface plasmon resonance(SPR) at 37° C. using a method such as that described in Example 2. Insome embodiments, the antibody or antigen-binding fragment thereof bindsa residue of human BTLA selected from: D52, P53, E55, E57, E83, Q86,E103, L106 and E92 as determined by x-ray crystallography or by flowcytometry of mutated receptors using a method such as that described inExample 5. In some embodiments, the antibody or antigen-binding fragmentthereof binds a residue of human BTLA selected from: Y39, K41, R42, Q43,E45 and S47. In some embodiments, the antibody or antigen-bindingfragment thereof binds a residue of human BTLA selected from: D35, T78,K81, S121 and L123. In some embodiments, the antibody or antigen-bindingfragment thereof binds residue H68 of BTLA. In some embodiments, theantibody or antigen-binding fragment thereof binds a residue of humanBTLA selected from: N65 and A64.

In particular embodiments of the first aspect of the invention, providedherein are isolated agonistic antibodies or antigen binding fragmentsthereof that specifically binds human B and T Lymphocyte Attenuator(BTLA) with an on off rate of less than 1×10⁻³ (1/s) as determined bysurface plasmon resonance (SPR) at 37° C. using a method such as thatdescribed in Example 2, wherein said antibody inhibits binding of BTLAto herpes virus entry mediator (HVEM) as determined by surface plasmonresonance (SPR) using a method such as that described in Example 4; andinhibits proliferation of T cells in vitro, as determined for example bya mixed lymphocyte reaction assay using a method such as that describedin Example 9. In some embodiments, said antibody or antigen bindingfragment thereof binds cynomolgus B and T Lymphocyte Attenuator (BTLA)with a KD of less than 10 nM, as determined by surface plasmon resonance(SPR) at 37° C. using a method such as that described in Example 2. Insome embodiments, said antibody or antigen binding fragment thereofbinds human B and T Lymphocyte Attenuator (BTLA) with a KD of less than2 nM, as determined by surface plasmon resonance (SPR) at 37° C. using amethod such as that described in Example 2. In some embodiments, theantibody or antigen-binding fragment thereof binds a residue of humanBTLA selected from: D52, P53, E55, E57, E83, Q86, E103, L106 and E92 asdetermined by x-ray crystallography or by flow cytometry of mutatedreceptors using a method such as that described in Example 5. In someembodiments, the antibody or antigen-binding fragment thereof binds aresidue of human BTLA selected from: Y39, K41, R42, Q43, E45 and S47. Insome embodiments, the antibody or antigen-binding fragment thereof bindsa residue of human BTLA selected from: D35, T78, K81, S121 and L123. Insome embodiments, the antibody or antigen-binding fragment thereof bindsresidue H68 of human BTLA. In some embodiments, the antibody orantigen-binding fragment thereof binds a residue of human BTLA selectedfrom: N65 and A64.

In particular embodiments of the first aspect of the invention, providedherein are isolated agonistic antibodies or antigen binding fragmentsthereof that specifically binds human B and T Lymphocyte Attenuator(BTLA), wherein said antibody binds cynomolgus BTLA with a KD of atleast 5 nM, as determined by surface plasmon resonance (SPR) at 37° C.using a method such as that described in Example 2; and wherein saidantibody inhibits binding of BTLA to herpes virus entry mediator (HVEM)as determined by surface plasmon resonance (SPR) using a method such asthat described in Example 4; and inhibits proliferation of T cells invitro, as determined for example by a mixed lymphocyte reaction assayusing a method such as that described in Example 9. In some embodiments,the antibody or antigen-binding fragment thereof binds a residue ofhuman BTLA selected from: D52, P53, E55, E57, E83, Q86, E103, L106 andE92 as determined by x-ray crystallography or by flow cytometry ofmutated receptors using a method such as that described in Example 5. Insome embodiments, the antibody or antigen-binding fragment thereof bindsa residue of human BTLA selected from: Y39, K41, R42, Q43, E45 and S47.In some embodiments, the antibody or antigen-binding fragment thereofbinds a residue of human BTLA selected from: D35, T78, K81, S121 andL123. In some embodiments, the antibody or antigen-binding fragmentthereof binds residue H68 of human BTLA. In some embodiments, theantibody or antigen-binding fragment thereof binds a residue of humanBTLA selected from: N65 andA64.

In particular embodiments of the first aspect of the invention, providedherein are isolated agonistic antibodies or antigen binding fragmentsthereof that specifically binds human B and T Lymphocyte Attenuator(BTLA), wherein said antibody binds cynomolgus BTLA with a KD of atleast than 50 nM, as determined by surface plasmon resonance (SPR) at37° C. using a method such as that described in Example 2; and whereinsaid antibody does not inhibit binding of BTLA to herpes virus entrymediator (HVEM) as determined by surface plasmon resonance (SPR) using amethod such as that described in Example 4; and inhibits proliferationof T cells in vitro, as determined for example by a mixed lymphocytereaction assay using a method such as that described in Example 9. Insome embodiments, the antibody or antigen-binding fragment thereof bindsa residue of human BTLA selected from: D52, P53, E55, E57, E83, Q86,E103, L106 and E92 as determined by x-ray crystallography or by flowcytometry of mutated receptors using a method such as that described inExample 5. In some embodiments, the antibody or antigen-binding fragmentthereof binds a residue of human BTLA selected from: Y39, K41, R42, Q43,E45 and S47. In some embodiments, the antibody or antigen-bindingfragment thereof binds a residue of human BTLA selected from: D35, T78,K81, S121 and L123. In some embodiments, the antibody or antigen-bindingfragment thereof binds residue H68 of human BTLA. In some embodiments,the antibody or antigen-binding fragment thereof binds a residue ofhuman BTLA selected from: N65 and A64.

In particular embodiments of the first aspect of the invention, providedherein are isolated agonistic antibodies or antigen binding fragmentsthereof that specifically binds human B and T Lymphocyte Attenuator(BTLA) with a KD from 1400 nM to 3500 nM, as determined by surfaceplasmon resonance (SPR) at 37° C. using a method such as that describedin Example 2; and wherein said antibody does not inhibit binding of BTLAto herpes virus entry mediator (HVEM) as determined by surface plasmonresonance (SPR) using a method such as that described in Example 4; andinhibits proliferation of T cells in in vitro, as determined for exampleby a mixed lymphocyte reaction assay using a method such as thatdescribed in Example 9. In some embodiments, said antibody or antigenbinding fragment thereof binds human BTLA with an on rate of at least2.0×10⁵ (1/Ms), as determined by surface plasmon resonance (SPR) at 37°C. using a method such as that described in Example 2. In someembodiments, said antibody or antigen binding fragment thereof bindshuman BTLA with an off rate of less than 10.0×10⁻¹ (1/s), as determinedby surface plasmon resonance (SPR) at 37° C. using a method such as thatdescribed in Example 2. In some embodiments, the antibody orantigen-binding fragment thereof binds a residue of human BTLA selectedfrom: D52, P53, E55, E57, E83, Q86, E103, L106 and E92 as determined byx-ray crystallography or by flow cytometry of mutated receptors using amethod such as that described in Example 5. In some embodiments, theantibody or antigen-binding fragment thereof binds a residue of humanBTLA selected from: Y39, K41, R42, Q43, E45 and S47. In someembodiments, the antibody or antigen-binding fragment thereof binds aresidue of human BTLA selected from: D35, T78, K81, S121 and L123. Insome embodiments, the antibody or antigen-binding fragment thereof bindsresidue H68 of human BTLA. In some embodiments, the antibody orantigen-binding fragment thereof binds a residue of human BTLA selectedfrom: N65 and A64.

In particular embodiments of the first aspect of the invention, providedherein are isolated agonistic antibodies or antigen binding fragmentsthereof that specifically binds human B and T Lymphocyte Attenuator(BTLA) with an on rate from 1.7×10⁵ (1/Ms) to 2.5×10⁵ (1/Ms), asdetermined by surface plasmon resonance (SPR) at 37° C. using a methodsuch as that described in Example 2; and wherein said antibody does notinhibit binding of BTLA to herpes virus entry mediator (HVEM) asdetermined by surface plasmon resonance (SPR) using a method such asthat described in Example 4; and inhibits proliferation of T cells in invitro, as determined for example by a mixed lymphocyte reaction assayusing a method such as that described in Example 9. In some embodiments,said antibody or antigen binding fragment thereof binds human BTLA withan off rate of less than 3.0×10⁻¹ (1/s), as determined by surfaceplasmon resonance (SPR) at 37° C. using a method such as that describedin Example 2. In some embodiments, said antibody or antigen bindingfragment thereof binds human BTLA with an off rate from 3.0×10⁻¹ (1/s)to 5.0×10⁻¹ (1/s), as determined by surface plasmon resonance (SPR) at37° C. using a method such as that described in Example 2. In someembodiments, said antibody or antigen binding fragment thereof bindshuman BTLA with a KD of at least 150 nM, as determined by surfaceplasmon resonance (SPR) at 37° C. using a method such as that describedin Example 2. In some embodiments, said antibody or antigen bindingfragment thereof binds human BTLA with a KD from 150 nM to 1500 nM, asdetermined by surface plasmon resonance (SPR) at 37° C. using a methodsuch as that described in Example 2. In some embodiments, said antibodyor antigen binding fragment thereof binds to an epitope that blocksbinding of 286 antibody. In some embodiments, the antibody orantigen-binding fragment thereof binds a residue of human BTLA selectedfrom: D52, P53, E55, E57, E83, Q86, E103, L106 and E92 as determined byx-ray crystallography or by flow cytometry of mutated receptors using amethod such as that described in Example 5. In some embodiments, theantibody or antigen-binding fragment thereof binds a residue of humanBTLA selected from: Y39, K41, R42, Q43, E45 and S47. In someembodiments, the antibody or antigen-binding fragment thereof binds aresidue of human BTLA selected from: D35, T78, K81, S121 and L123. Insome embodiments, the antibody or antigen-binding fragment thereof bindsresidue H68 of human BTLA. In some embodiments, the antibody orantigen-binding fragment thereof binds a residue of human BTLA selectedfrom: N65 and A64.

In particular embodiments of the first aspect of the invention, providedherein are isolated agonistic antibodies or antigen binding fragmentsthereof that specifically binds human B and T Lymphocyte Attenuator(BTLA) with a KD from 40nM to 1200nM, as determined by surface plasmonresonance (SPR) at 37° C. using a method such as that described inExample 2; and wherein said antibody does not inhibit binding of BTLA toherpes virus entry mediator (HVEM) as determined by surface plasmonresonance (SPR) using a method such as that described in Example 4; andinhibits proliferation of T cells in in vitro, as determined for exampleby a mixed lymphocyte reaction assay using a method such as thatdescribed in Example 9. In some embodiments, said antibody or antigenbinding fragment thereof binds human BTLA with an on rate of at least1.0×10⁵ (1/Ms), as determined by surface plasmon resonance (SPR) at 37°C. using a method such as that described in Example 2. In someembodiments, said antibody or antigen binding fragment thereof bindshuman BTLA with an on rate from 1.0×10⁵ (1/Ms) to 10×10⁵ (1/Ms), asdetermined by surface plasmon resonance (SPR) at 37° C. using a methodsuch as that described in Example 2. In some embodiments, said antibodyor antigen binding fragment thereof binds human BTLA with an off rate ofless than 6.0×10⁻¹ (1/s), as determined by surface plasmon resonance(SPR) at 37° C. using a method such as that described in Example 2. Insome embodiments, said antibody or antigen binding fragment thereofbinds human BTLA with an off rate from 6.0×10⁻¹ (1/s) to 10.0×10⁻²(1/s),as determined by surface plasmon resonance (SPR) at 37° C. using amethod such as that described in Example 2. In some embodiments, theantibody or antigen-binding fragment thereof binds a residue of humanBTLA selected from: D52, P53, E55, E57, E83, Q86, E103, L106 and E92 asdetermined by x-ray crystallography or by flow cytometry of mutatedreceptors using a method such as that described in Example 5. In someembodiments, the antibody or antigen-binding fragment thereof binds aresidue of human BTLA selected from: Y39, K41, R42, Q43, E45 and S47. Insome embodiments, the antibody or antigen-binding fragment thereof bindsa residue of human BTLA selected from: D35, T78, K81, S121 and L123. Insome embodiments, the antibody or antigen-binding fragment thereof bindsresidue H68 of human BTLA. In some embodiments, the antibody orantigen-binding fragment thereof binds a residue of human BTLA selectedfrom: N65 and A64.

In certain embodiments the isolated antibody or an antigen-bindingfragment thereof of the invention that specifically binds human BTLAincreases BTLA activity and/or signaling through the receptor.

In particular embodiments, the antibody of the invention is selectedfrom the group consisting of: a human antibody, a humanised antibody, achimeric antibody, a multispecific antibody (such as a bispecificantibody).

In particular embodiments, the antigen-binding fragment of the inventionis selected from the group consisting of: scFv, sc(Fv)², dsFv, Fab,Fab′, (Fab′)2 and diabody.

In particular embodiments, the heavy chain and light chain moleculesthat form the antigen-binding fragment are connected by a flexiblelinker. There are many commonly used flexible linkers and the choice oflinker can be made by a person of skill in the art.

The peptide linker connecting scFv VH and VL domains joins the carboxylterminus of one variable region domain to the amino terminus of anothervariable domain without significantly compromising the fidelity of theVH-VL pairing and antigen-binding sites. Peptide linkers can vary from10 to 25 amino acids in length and are typically, but not always,composed of hydrophilic amino acids such as glycine (G) and serine (S).The linker can be one that is found in natural multi-domain proteins(e.g. see Argos P. J Mol Biol. 211:943-958, 1990; and. Heringa G.Protein Eng. 15:871-879, 2002), or adapted therefrom.

Commonly used flexible linkers have sequences consisting primarily ofstretches of Gly and Ser residues (“GS” linker). An example of the mostwidely used flexible linker has the sequence of (Gly-Gly-Gly-Gly-Ser)n.By adjusting the copy number “n”, the length of this GS linker can bealtered to achieve appropriate separation of the functional domains, orto maintain necessary inter-domain interactions. Generally, the (GGGGS)3peptide is used as an scFv peptide linker (Leith et al., Int. J. Oncol.24:765-771, 2004; Holiger et al. Proc. Natl. Acad. Sci. U.S.A.90:6444-6448, 1993). This 15-amino acid linker sequence [designated asthe (GGGGS)3 linker] is used in the Recombinant Phage Antibody System(RPAS kit) commercially available from Amersham. Several other linkershave also been used to create scFV molecules (e.g. KESGSVSSEQLAQFRSLDand EGKSSGSGSESKST; Bird et al., Science 242:432-426, 1988).

The inventors have mapped the epitopes on BTLA where the potent 2.8.6and 11.5.1 agonist and the other antibodies disclosed herein bind.

In particular embodiments, the antibody or antigen-binding fragmentthereof of the invention binds a residue of human BTLA selected from:D52, P53, E55, E57, E83, Q86, E103, L106, E92, Y39, K41, R42, Q43, E45,S47, D35, T78, K81, 5121, L123, H68, N65, A64.

In particular embodiments, the antibody or antigen-binding fragmentthereof of the invention binds a residue of human BTLA selected from:D52, P53, E55, E57, E83, Q86, E103, L106, E92.

In particular embodiments, the antibody or antigen-binding fragmentthereof of the invention binds at least two residues of human BTLAselected from: D52, P53, E55, E57, E83, Q86, E103, L106, E92.

In particular embodiments, the antibody or antigen-binding fragmentthereof of the invention binds at least three residues of human BTLAselected from: D52, P53, E55, E57, E83, Q86, E103, L106 and E92.

In particular embodiments, the antibody or antigen-binding fragmentthereof of the invention binds at least five residues of human BTLAselected from: D52, P53, E55, E57, E83, Q86, E103, L106 and E92.

In particular embodiments, the antibody or antigen-binding fragmentthereof of the invention binds all of the residues of human BTLAselected from: D52, P53, E55, E57, E83, Q86, E103, L106 and E92.

In particular embodiments, the antibody or antigen-binding fragmentthereof of the invention binds a residue of human BTLA selected from:Y39, K41, R42, Q43, E45 and S47.

In a particular embodiment, the antibody or antigen-binding fragmentthereof of the invention binds at least two residues of human BTLAselected from: Y39, K41, R42, Q43, E45 and S47.

In particular embodiments, the antibody or antigen-binding fragmentthereof of the invention binds all of the residues of human BTLAselected from: Y39, K41, R42, Q43, E45 and S47.

In particular embodiments, the antibody or antigen-binding fragmentthereof of the invention binds a residue of human BTLA selected from:D35, T78, K81, S121and L123.

In a particular embodiment, the antibody or antigen-binding fragmentthereof of the invention binds at least two residues of human BTLAselected from: D35, T78, K81, S121 and L123.

In particular embodiments, the antibody or antigen-binding fragmentthereof of the invention binds residue H68 of human BTLA

In particular embodiments, the antibody or antigen-binding fragmentthereof of the invention binds a residue of human BTLA selected from:N65 and A64.

In particular embodiments, the antibody or antigen-binding fragmentthereof of the invention binds both the N65 and A64 residues of humanBTLA.

The numbering of the residues, such as K41 refers to the amino acid (K;lysine) at position 41; wherein the numbering refers to the position inhuman BTLA polypeptide as disclosed in SEQ ID NO: 23.

In particular embodiments, the antibody of the invention is an IgG1,IgG2 or IgG4 antibody. In particular embodiments the antibody is amurine or human antibody.

In a particular embodiment, the antibody or antigen-binding fragmentthereof of the invention is a humanised antibody.

In a particular embodiment, the antibody or antigen-binding fragmentthereof of the invention is a fully human antibody.

In a particular embodiment, the antibody or antigen-binding fragmentthereof of the invention acts as an agonist inducing signaling throughthe BTLA receptor.

The antibodies (or antigen-binding fragments thereof) of the inventionare particularly potent agonists.

In a particular embodiment, the antibody or antigen-binding fragmentthereof of the invention have EC50s of not more than 1 nM.

The agonist antibodies (or antigen-binding fragments thereof) of theinvention have particularly high efficacy.

In a particular embodiment the antibody or antigen-binding fragmentthereof of the invention inhibits T cell proliferation by at least 20%,suitably by at least 30%, more suitably by at least 40%.

In a particular embodiment the antibody or antigen-binding fragmentthereof of the invention inhibits T cell IFN-gamma production by atleast 50%, suitably by at least 75%, more suitably by at least 95%, asmeasured for example by ELISA of supernatants in an in vitro mixedlymphocyte reaction.

In a particular embodiment the antibody or antigen-binding fragmentthereof of the invention inhibits T cell IL-2 production by at least50%, suitably by at least 75%, more suitably by at least 95%, asmeasured for example by ELISA of supernatants in an in vitro mixedlymphocyte reaction.

In a particular embodiment the antibody or antigen-binding fragmentthereof of the invention inhibits T cell IL-17 production by at least50%, suitably by at least 75%, more suitably by at least 95%, asmeasured for example by ELISA of supernatants in an in vitro mixedlymphocyte reaction.

In a particular embodiment the antibody or antigen-binding fragmentthereof of the invention reduces mortality in a murine GVHD model by atleast 50%, suitably by at least 75%, more suitably by at least 95%,using a method such as that described in Example 12.

In a particular embodiment the antibody or antigen-binding fragmentthereof of the invention reduces weight loss in a murine T-cell colitismodel by at least 50%, suitably by at least 75%, more suitably by atleast 95%, using a method such as that described in Example 11.

In a particular embodiment the antibody or antigen-binding fragmentthereof of the invention reduces colon inflammation in a murine T-cellcolitis model by at least 50%, suitably by at least 75%, more suitablyby at least 95%, using a method such as that described in Example 11.

In certain aspects, the invention also relates to an isolatedpolypeptide comprising the VL domains or the VH domains of any of theantibodies or antigen-binding fragments thereof described herein.

In a particular embodiment, the isolated polypeptide comprises an aminoacid sequence as set forth in SEQ ID NO: 13 or 14, or a sequence with atleast 90% identity thereto.

Nucleic Acid Molecules

The antibody or antigen-binding fragment thereof of the invention willbe encoded by nucleic acid. The antibody or antigen-binding fragmentthereof may be encoded by a single nucleic acid molecule or it may beencoded by two or more nucleic acid molecules. For example, as theantigen binding site is typically formed by the coming together of aheavy chain variable polypeptide region and a light chain variablepolypeptide region, the two variable (heavy and light) polypeptideregions may be encoded by separate nucleic acid molecules.Alternatively, for example in the case of an ScFv, they may be encodedby the same nucleic acid molecule.

According to a second aspect of the invention there is provided one ormore nucleic acid molecules that encode an antibody or antigen-bindingfragment thereof in accordance with the first aspect of the invention.

From the primary amino acid sequence of the polypeptide(s) encoding anantibody or antigen-binding fragment thereof of the invention the personof skill in the art is able to determine suitable nucleotide sequence(s)that encodes the polypeptide(s) and, if desired, one that iscodon-optimised (e.g. see Mauro and Chappell. Trends Mol Med.20(11):604-613, 2014).

As used herein, when there is reference to a previous aspect of theinvention, e.g. “in accordance with the first (or second etc.) aspect ofthe invention”, it is understood to also cover any recited variation ofsaid aspect (e.g. variation of the first (or second etc.) aspect).

According to a variation of the second aspect of the invention there isprovided an isolated nucleic acid comprising a nucleotide sequence thatencodes a heavy chain variable region polypeptide or a light chainvariable region polypeptide of the invention. A heavy chain variablepolypeptide or a light chain variable polypeptide of the inventionrefers to the individual polypeptide chains that include amino acidsthat make up part of the antigen-binding site. Of course, the saidpolypeptides may also comprise other domains such as constant domains,hinge regions, and an Fc region, such as one comprising one or more Fcreceptor binding sites.

According to another variation of the second aspect of the inventionthere is provided an isolated nucleic acid which comprises one or morenucleotide sequence encoding polypeptides capable of forming an antibodyor antigen-binding fragment of the invention. In particular embodiments,the said polypeptides may also comprise other domains such as constantdomains, hinge regions, and an Fc region, such as one comprising one ormore Fc receptor binding sites.

One of the nucleic acid molecules may encode just the polypeptidesequence that comprises the VL domain of the antibody or fragmentthereof. One of the nucleic acid molecules may encode just thepolypeptide sequence that comprises the VH domain of the antibody orfragment thereof. However, the nucleic acid molecule may also encodeboth VH and VL domain containing polypeptide sequences capable offorming the antibody or antibody fragment thereof of the invention.

The nucleic acid molecule(s) that encode the antibody or antigen-bindingfragment thereof of the invention, such as according to the first aspectof the invention, may be, or may be part of, a vector (such as a plasmidvector, cosmid vector or viral vector, or an artificial chromosome) thatmay comprise other functional regions (elements) such as one or morepromoters, one or more origins or replication, one or more selectablemarker(s), and one or more other elements typically found in expressionvectors. The cloning and expression of nucleic acids that encodeproteins, including antibodies, is well established and well within theskill of the person in the art.

According to a third aspect of the invention there is provided a vectorcomprising the nucleic acid of the second aspect of the invention. Inparticular embodiments, the vector is a plasmid vector, cosmid vector,viral vector, or an artificial chromosome.

The nucleic acids of the invention, including vector nucleic acids thatcomprise nucleotide sequences that encode the polypeptides capable offorming an antibody of the invention or an antigen-binding fragmentsthereof, may be in purified/isolated form.

Isolated/purified nucleic acids that encode an antibody orantigen-binding fragment thereof of the invention will be free orsubstantially free of material with which they are naturally associated,such as other proteins or nucleic acids with which they are found intheir natural environment, or the environment in which they are prepared(e.g. cell culture) when such preparation is by recombinant DNAtechnology practised in vitro or in vivo.

In particular embodiments, the nucleic acids of the invention aregreater than 80%, such as greater than 90%, greater than 95%, greaterthan 97% and greater than 99% pure.

Thus, according to another variation of the third aspect of theinvention there is provided a vector comprising a nucleic acid ornucleotide sequence that encodes a heavy chain variable polypeptide or alight chain variable polypeptide of the invention. In a particularembodiment, the vector comprises nucleic acid that encodes both theheavy and light chain variable regions. In particular embodiments, thesaid polypeptides may also comprise other domains such as constantdomains, hinge regions, and an Fc region, such as one comprising one ormore Fc receptor binding sites.

The nucleic acid and/or vector of the invention may be introduced into ahost cell. The introduction may employ any available technique. Foreukaryotic cells, suitable techniques may include calcium phosphatetransfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. Introducing nucleic acid inthe host cell, in particular a eukaryotic cell may use a viral or aplasmid-based system. The plasmid system may be maintained episomally ormay incorporated into the host cell or into an artificial chromosome.Incorporation may be either by random or targeted integration of one ormore copies at single or multiple loci. For bacterial cells, suitabletechniques may include calcium chloride transformation, electroporationand transfection using bacteriophage.

In one embodiment, the nucleic acid of the invention is integrated intothe genome (e.g. chromosome) of the host cell. Integration may bepromoted by inclusion of sequences that promote recombination with thegenome, in accordance with standard techniques.

Host Cells

A further aspect of the present invention provides a host cellcontaining nucleic acid as disclosed herein. Such a host cell may be invitro and may be in culture.

The host cell can be from any species, such as a bacterium or yeast butsuitably the host cell is an mammalian cell such as a human cell orrodent cell, for example a HEK293T cell or CHO-K1 cell.

Thus, according to a fourth aspect of the invention there is provided ahost cell comprising the nucleic acid sequence according to the secondaspect of the invention or the vector according to third aspect of theinvention.

The host cell can be treated so as to cause or allow expression of theprotein of the invention from the nucleic acid, e.g. by culturing hostcells under conditions for expression of the encoding nucleic acid. Thepurification of the expressed product may be achieved by methods knownto one of skill in the art.

Thus, the nucleic acids of the invention, including vector nucleic acidsthat comprise nucleotide sequences that encode the polypeptides capableof forming the antibodies of the invention or antigen-binding fragmentsthereof, may be present in an isolated host cell. The host cell istypically part of a clonal population of host cells. As used herein,reference to a host cell also encompasses a clonal population of saidcell. A clonal population is one that has been grown from a singleparent host cell. The host cell can be from any suitable organism.Suitable host cells include bacterial, fungal or mammalian cells.

The host cell may serve to assist in amplifying the vector nucleic acid(such as with a plasmid) or it may serve as the biological factory toexpress the polypeptide(s) of the invention that form the BTLA antibodyor antigen-binding fragment thereof of the invention. A suitable hostfor amplifying the vector nucleic acid could be a bacterial or fungalcell, such as an Escherichia coli cell or Saccharomyces cerevisiae cell.A suitable host for expressing the proteins of the invention (i.e. thepolypeptides making up the human BTLA-binding antibody orantigen-binding fragment thereof of the invention would be a mammaliancell such as a HEK293T or CHO-K1 cell. In a particular embodiment, thehost cell is a mammalian cell, such as a HEK293T or CHO-K1 cell.

A variety of host-expression vector systems may be utilized to express aBTLA-binding molecule as described herein (see e.g. U.S. Pat. No.5,807,715). For example, mammalian cells such as Chinese hamster ovarycells (CHO), in conjunction with a vector such as the major intermediateearly gene promoter element from human cytomegalovirus is an effectiveexpression system for CEA proteins (Foecking et al., Gene, 45:101(1986); and Cockett et al., Bio/Technology, 8:2 (1990)). Different hostcells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the protein of thedisclosure. To this end, eukaryotic host cells which possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Suchmammalian host cells include but are not limited to CHO, HEK, VERY, BHK,Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D,NS0, CRL7O3O and HsS78Bst cells.

Antibody Production

According to a fifth aspect of the invention there is provided a methodof producing an antibody or an antigen-binding fragment thereofaccording to the first aspect of the invention, comprising the step ofculturing the host cell of the fourth aspect of the invention underconditions for production of said an antibody or antigen-bindingfragment thereof, and optionally isolating and/or purifying saidantibody or antigen-binding fragment thereof.

According to a variation of the fifth aspect of the invention there isprovided a method of producing an antibody or an antigen-bindingfragment thereof that binds to human BTLA, comprising the step ofculturing the host cell that comprises nucleic acid encoding thepolypeptide(s) that form the antibody or the antigen-binding fragmentthereof that binds to human BTLA under conditions for production of saidantibody or antigen-binding fragment thereof, optionally furthercomprising isolating/purifying said antibody or antigen-binding fragmentthereof.

By isolated/purified we mean that the antibody or antigen-bindingfragment thereof of the invention, or polypeptides that make up thesemolecules, will be free or substantially free of material with whichthey are naturally associated, such as other proteins or nucleic acidswith which they are found in their natural environment, or theenvironment in which they are prepared (e.g. cell culture) when suchpreparation is by recombinant DNA technology practised in vitro or invivo.

According to a variation of the fifth of the invention there is provideda method for preparing an antibody or antigen-binding fragment thereofthat specifically binds human BTLA, the method comprising the steps of:

a) providing a host cell comprising one or more nucleic acid moleculesencoding the amino acid sequence of a heavy chain variable domain and alight chain variable domain which when expressed are capable ofcombining to create a human BTLA-binding molecule;

b) culturing the host cell expressing the encoded amino acid sequence;and c) isolating the antibody or antigen-binding fragment molecule.

The one or more nucleic acid molecules are those describe above thatencode for polypeptides capable of forming an antibody orantigen-binding fragment thereof of the invention that specificallybinds human BTLA.

In a particular embodiment, the antibody or antigen-binding fragmentthereof comprises: i) a heavy chain variable region comprising threeCDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1 has an amino acid sequenceas set forth in SEQ ID NO: 1, CDRH2 has an amino acid sequence as setforth in SEQ ID NO: 2, and CDRH3 has an amino acid sequence as set forthin SEQ ID NO: 3; and

ii) a light chain variable region comprising three CDRs: CDRL1, CDRL2and CDRL3, wherein CDRL1 has an amino acid sequence as set forth in SEQID NO: 4, CDRL2 has an amino acid sequence as set forth in SEQ ID NO: 5,and CDRL3 has an amino acid sequence as set forth in SEQ ID NO: 6.

In a particular embodiment, the antibody or antigen-binding fragmentthereof comprises: i) a heavy chain variable region comprising threeCDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1 has an amino acid sequenceas set forth in SEQ ID NO: 7, CDRH2 has an amino acid sequence as setforth in SEQ ID NO: 8, and CDRH3 has an amino acid sequence as set forthin SEQ ID NO: 9; and

ii) a light chain variable region comprising three CDRs: CDRL1, CDRL2and CDRL3, wherein CDRL1 has an amino acid sequence as set forth in SEQID NO: 10, CDRL2 has an amino acid sequence as set forth in SEQ ID NO:11, and CDRL3 has an amino acid sequence as set forth in SEQ ID NO: 12.

In a particular embodiment, the antibody or antigen-binding fragmentthereof comprises:

i) a heavy chain variable region comprising an amino acid sequencedisclosed in SEQ ID NO: 13, or a sequence with at least 90% sequenceidentity thereto; and

ii) a light chain variable region comprising an amino acid sequencedisclosed in SEQ ID NO:14, or a sequence with at least 90% sequenceidentity thereto.

In various embodiments, the antibody or antigen-binding fragment thereofcomprises a heavy chain and a light chain, wherein the heavy chaincomprises a heavy chain variable region comprising three CDRs: CDRH1,CDRH2 and CDRH3, and the light chain comprises a light chain variableregion comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein (1) CDRH1,CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 31,SEQ ID NO: 32, and SEQ ID NO: 33, respectively, and CDRL1, CDRL2, andCDRL3 have an amino acid sequence as set forth in SEQ ID NO: 34, SEQ IDNO: 35, and SEQ ID NO: 36, respectively; (2) CDRH1, CDRH2, CDRH3 have anamino acid sequence as set forth in SEQ ID NO: 37, SEQ ID NO: 38, andSEQ ID NO: 39, respectively, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ IDNO: 42, respectively; (3) CDRH1, CDRH2, CDRH3 have an amino acidsequence as set forth in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO:45, respectively, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO:48, respectively; (4) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 43, SEQ ID NO: 56, and SEQ ID NO: 57,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60,respectively; (5) CDRH1, CDRH2, CDRH3 have an amino acid sequence as setforth in SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63, respectively,and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth inSEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; (6)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 31, SEQ ID NO: 32, and SEQ ID NO: 69, respectively, and CDRL1,CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO:34, SEQ ID NO: 35, and SEQ ID NO: 72, respectively; (7) CDRH1, CDRH2,CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 73, SEQ IDNO: 74, and SEQ ID NO: 75, respectively, and CDRL1, CDRL2, and CDRL3have an amino acid sequence as set forth in SEQ ID NO: 76, SEQ ID NO:47, and SEQ ID NO: 78, respectively; (8) CDRH1, CDRH2, CDRH3 have anamino acid sequence as set forth in SEQ ID NO: 79, SEQ ID NO: 80, andSEQ ID NO: 81, respectively, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 82, SEQ ID NO: 83, and SEQ IDNO: 84, respectively; (9) CDRH1, CDRH2, CDRH3 have an amino acidsequence as set forth in SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO:87, respectively, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO:90, respectively; (10) CDRH1, CDRH2, CDRH3 have an amino acid sequenceas set forth in SEQ ID NO: 61, SEQ ID NO: 92, and SEQ ID NO: 93,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96,respectively; (11) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102,respectively; (12) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 64, SEQ ID NO: 107, and SEQ ID NO: 108,respectively; (13) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 114,respectively; (14) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 85, SEQ ID NO: 110, and SEQ ID NO: 117,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 114,respectively; (15) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 126,respectively; (16) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36,respectively; (17) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 136, SEQ ID NO: 137, and SEQ ID NO: 138,respectively; (18) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 139, SEQ ID NO: 140, and SEQ ID NO: 141,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 144,respectively; (19) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 145, SEQ ID NO: 146, and SEQ ID NO: 147,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150,respectively; (20) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO: 150,respectively; (21) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 159,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 136, SEQ ID NO: 137, and SEQ ID NO: 162,respectively; (22) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 169, SEQ ID NO: 170, and SEQ ID NO: 171,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 172, SEQ ID NO: 173, and SEQ ID NO: 174,respectively; (23) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 180,respectively; (24) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 31, SEQ ID NO: 182, and SEQ ID NO: 183,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 136, SEQ ID NO: 137, and SEQ ID NO: 186,respectively; (25) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 187, SEQ ID NO: 188, and SEQ ID NO: 189,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 190, SEQ ID NO: 191, and SEQ ID NO: 192,respectively; (26) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 193, SEQ ID NO: 194, and SEQ ID NO: 195,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 196, SEQ ID NO: 197, and SEQ ID NO: 12,respectively; (27) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 199, SEQ ID NO: 200, and SEQ ID NO: 201,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 202, SEQ ID NO: 203, and SEQ ID NO: 6,respectively; (28) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 142, SEQ ID NO: 209, and SEQ ID NO: 210,respectively; (29) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 211, SEQ ID NO: 212, and SEQ ID NO: 213,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 214, SEQ ID NO: 35, and SEQ ID NO: 216,respectively; (30) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 127, SEQ ID NO: 386, and SEQ ID NO: 129,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36,respectively; (31) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 205, SEQ ID NO: 206, and SEQ ID NO: 207,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 142, SEQ ID NO: 209, and SEQ ID NO: 210,respectively; (32) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 127, SEQ ID NO: 388, and SEQ ID NO: 129,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36,respectively; (33) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 205, SEQ ID NO: 387, and SEQ ID NO: 207,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 142, SEQ ID NO: 209, and SEQ ID NO: 210,respectively; or (34) CDRH1, CDRH2, CDRH3 have an amino acid sequence asset forth in SEQ ID NO: 205, SEQ ID NO: 387, and SEQ ID NO: 207,respectively, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence asset forth in SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 210,respectively; wherein from 0 to 3 amino acid modifications can bepresent in any CDR/SEQ ID NO:

Conditions for the production of the antibody or an antigen-bindingfragment thereof of the invention and purification of said molecules arewell-known in the art.

One way of attending to this is to prepare a clonal population of cellscapable of expressing the antibody or fragment thereof of the inventionand culturing these in a suitable growth medium for a period of time andat a temperature conducive to allow for expansion/growth of the cellpopulation and expression of the protein(s) of interest. If theprotein(s) of interest (e.g. antibody of invention) is expressed withinthe host cells then the cells may be lysed (e.g. using a mild detergentor sonication) to release the contents of the cell (and thus the proteinof interest) into the surrounding medium (which could be the culturemedium or another medium that the cells have been reconstituted in) andthis medium is then subjected to purification processes. If theprotein(s) of interest (e.g. antibody of invention) is secreted into thegrowth medium, then the medium is subjected to purification processes.Antibody purification typically involves isolation of antibody from, forexample the medium or from the culture supernatant of a hybridoma cellline using well-established methods typically involving chromatography(e.g., using affinity chromatography, anionic and/or cationic exchangechromatography, size-exclusion chromatography or other separationtechniques) to separate the protein of interest from unwantedhost-derived proteins and other cellular contaminants (e.g. nucleicacids, carbohydrates etc.). The purified proteins may also be subjectedto a virus inactivation step. Finally, the purified protein of interestmay, for example, be lyophilised or formulated ready for storage,shipment and subsequent use. Preferably the protein of interest (e.g.antibody or antigen-binding fragment thereof of the invention) will besubstantially free from contaminating proteins which were originallypresent in the culture medium following expression or cell-lysis.

In certain embodiments, the antibody or antigen-binding fragment thereofof the invention will be at least 90%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% pure.

The proteins of the invention (antibody or antigen-binding fragmentthereof of the invention) can be formulated into a suitable composition.

Compositions

While the BTLA-binding molecule may be administered alone, in certainembodiments administration is of a pharmaceutical composition whereinthe BTLA-binding molecule is formulated with at least onepharmaceutically-acceptable excipient. The excipient may be a suitablepharmaceutical carrier solute. Such carriers are well known in the artand include phosphate buffered saline solutions, water, liposomes,various types of wetting agents, sterile solutions, etc. Compositionscomprising such carriers can be formulated by well-known conventionalmethods. These pharmaceutical compositions can be administered to thesubject at a suitable dose. The dosage regimen will be determined by theattending physician and clinical factors.

According to a sixth aspect of the invention there is provided apharmaceutical composition comprising a pharmaceutically acceptableexcipient and a therapeutically effective amount of the antibody orantigen-binding fragment thereof of the first aspect of the invention,or that produced by the fifth aspect of the invention. In a particularembodiment, the composition comprises phosphate buffered saline.

A “pharmaceutical composition” refers to a preparation which is in suchform as to permit the biological activity of the active ingredient to beeffective, and which contains no additional components which areunacceptably toxic to a subject to which the formulation would beadministered. The pharmaceutical composition will include one or morepharmaceutically acceptable excipients. The term excipient in thiscontext refers to any additive, such as fillers, solubilisers, carriers,vehicles, additives and the like.

The pharmaceutical compositions can comprise one or morepharmaceutically acceptable excipients, including, e.g., water, ionexchangers, proteins, buffer substances, and salts. Preservatives andother additives can also be present. The excipient can be a solvent ordispersion medium. Suitable formulations for use in therapeutic methodsdisclosed herein are described in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

“Pharmaceutically acceptable” excipients are those which can reasonablybe administered to a subject mammal to provide an effective dose of theactive ingredient employed. Pharmaceutical compositions of the inventionare prepared for storage by mixing the composition with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptableexcipients are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN(TM),PLURONICS(TM) or polyethylene glycol (PEG). Lyophilized HER2 antibodyformulations are described in WO 97/04801.

The pharmaceutical compositions to be used for in vivo administrationmust be sterile. This can be readily accomplished by filtration throughsterile filtration membranes.

The route of administration of the BTLA binding moiety molecule, e.g.,an antibody, or antigen-binding fragment thereof can be, for example,oral, parenteral, by inhalation or topical. The term parenteral as usedherein includes, e.g., intravenous, intraarterial, intraperitoneal,intramuscular, subcutaneous, rectal, or vaginal administration.

Pharmaceutical compositions for parenteral administration includesterile aqueous or non-aqueous solutions, and suspensions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol, andinjectable organic esters such as ethyl oleate. Aqueous carriers includewater, aqueous solutions, or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishes,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. In addition, the composition might compriseproteinaceous carriers, like, e.g., serum albumin or immunoglobulin, incertain embodiments of human origin. For intravenous injection, orinjection at the site of affliction, the active ingredient will be inthe form of a parenterally acceptable aqueous solution which ispyrogen-free and has suitable pH, isotonicity and stability. Those ofrelevant skill in the art are well able to prepare suitable solutionsusing, for example, isotonic vehicles such as Sodium Chloride Injection,Ringer's Injection, Lactated Ringer's Injection. Preservatives,stabilisers, buffers, antioxidants and/or other additives may beincluded, as required. As noted above, these are all referred to hereinas excipients.

Compositions for injection can be administered with medical devicesknown in the art. For example, with a hypodermic needle. Needlelessinjection devices, such as those disclosed in U.S. Pat. Nos: 6,620,135and 5,312,335 could also be utilised.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder, liquid or semi-solid form. A tablet may comprise asolid carrier such as gelatin or an adjuvant. Liquid pharmaceuticalcompositions generally comprise a liquid carrier such as water,petroleum, animal or vegetable oils, mineral oil or synthetic oil.Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolmay be included as required.

An antibody or antigen-binding fragment thereof of the present inventionmay be formulated in liquid, semi-solid or solid forms depending on thephysicochemical properties of the molecule and the route of delivery.Formulations may include excipients, or combinations of excipients, forexample: sugars, amino acids and surfactants. Liquid formulations mayinclude a wide range of antibody concentrations and pH. Solidformulations may be produced by lyophilisation, spray drying, or dryingby supercritical fluid technology, for example.

The pharmaceutical composition can be administered as a single dose,multiple doses or over an established period of time in an infusion.Dosage regimens also can be adjusted to provide the optimum desiredresponse (e.g., a therapeutic or prophylactic response). In particular,parenteral formulations can be a single bolus dose, an infusion or aloading bolus dose followed with one or more maintenance doses. Thesecompositions can be administered at specific fixed or variableintervals, e.g., once a day, or on an “as needed” basis.

Dosages

The amount of the BTLA-binding molecule, or the pharmaceuticalformulation containing such molecule, which will be therapeuticallyeffective can be determined by standard clinical techniques, such asthrough dose ranging clinical trials. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.The dosage of the compositions to be administered can be determined bythe skilled artisan without undue experimentation in conjunction withstandard dose-response studies. Relevant circumstances to be consideredin making those determinations include the condition or conditions to betreated, the choice of composition to be administered, the age, weight,and response of the individual patient, and the severity of thepatient's symptoms. For example, the actual patient body weight may beused to calculate the dose of the formulations in milliliters (mL) to beadministered. There may be no downward adjustment to “ideal” weight. Insuch a situation, an appropriate dose may be calculated by the followingformula:

Dose(ml.)=[patient weight (kg)×dose level (mg/kg)/drug concentration(mg/mL)]

Therapeutically effective doses of the pharmaceutical compositions forthe treatment of BTLA-related diseases or disorders, as discussedherein, will vary depending upon many different factors, including meansof administration, target site, physiological state of the patient,weight or patient, sex of patient, age of patient, whether the patientis human or an animal, other medications administered, and whethertreatment is prophylactic or therapeutic. The therapeutically effectivedose is likely to have been determined from clinical trials and issomething that the attending physician can determine using treatmentguidelines. Usually, the patient is a human, but non-human mammals canalso be treated. Treatment dosages can be titrated using routine methodsknown to those of skill in the art to optimize safety and efficacy.

In various embodiments, the BTLA-binding molecule is administered at aconcentration of about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg,about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17 mg/kg, about 18mg/kg, about 19 mg/kg, or about 20 mg/kg.

A pharmaceutical composition of the invention may be administered aloneor in combination with other treatments, either simultaneously orsequentially dependent upon the condition to be treated. Suchcombination would likely be with other immunosuppressives such as oneselected from: corticosteroids, cyclosporine, azathioprine,sulfasalazine, methotrexate, mycophenolate, tacrolimus and fingolimod,or other biologics such as infliximab, adalimumab, ustekinumab,tocilizumab and rituximab.

According to a seventh aspect of the invention there is provided amethod of preparing a pharmaceutical composition, the method comprisingformulating an antibody or an antigen-binding fragment thereof inaccordance with the first aspect of the invention, or one produced inaccordance with the fifth aspect of the invention into a compositionincluding at least one additional component. In a particular embodiment,the at least one additional component is a pharmaceutically acceptableexcipient.

Kits

Further, the product (e.g. BTLA binding molecule or a pharmaceuticalcomposition thereof) can be packaged and sold in the form of a kit. Sucharticles of manufacture can have labels or package inserts indicatinginstructions about the product and the appropriate use of the productfor the treatment of a subject suffering from or predisposed to adisease or disorder.

Thus, according to one aspect of the invention there is provided a kitcomprising an antibody or antigen-binding fragment thereof in accordancewith the first aspect of the invention or the pharmaceutical compositionin accordance with the sixth aspect of the invention. Suitably, such akit includes a package insert comprising instructions for use.

Therapy/Medical Uses

An antibody or antigen-binding fragment thereof of the invention or apharmaceutical composition comprising said antibody or antigen-bindingfragment thereof may be used in therapy, typically as a medicament.

In certain embodiments, an antibody or antigen-binding fragment thereofof the invention or a pharmaceutical composition comprising saidantibody or antigen-binding fragment thereof may be used for treating orpreventing any disease or condition in a subject in need thereof.

BTLA is involved in down-regulating immune responses and there are manydiseases or conditions that could be treated by suppressing host T-cellsand/or B-cells (e.g. see Crawford & Wherry. Editorial: Therapeuticpotential of targeting BTLA. J Leukocyte Biol. 86:5-8, 2009). Diseasesor conditions that could benefit from treatment with an anti-BTLAagonist are referred to herein as “BTLA-related diseases”. BTLA-relateddiseases include inflammatory or autoimmune diseases, and disorders ofexcessive immune cell proliferation.

Specific BTLA-related diseases that can be treated with the BTLA-bindingmolecules of the invention include: Addison's disease, allergy, alopeciaareata, amyotrophic lateral sclerosis, ankylosing spondylitis,anti-phospholipid syndrome, asthma (including allergic asthma),autoimmune haemolytic anaemia, autoimmune hepatitis, autoimmunepancreatitis, autoimmune polyendocrine syndrome, Behcet's disease,bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinatingpolyneuropathy, coeliac disease, Crohn's disease, Cushing's Syndrome,dermatomyositis, diabetes mellitus type 1, eosinophilic granulomatosiswith polyangiitis, graft versus host disease, Graves' disease,Guillain-Barre syndrome, Hashimoto's thyroiditis, HidradenitisSuppurativa, inflammatory fibrosis (e.g., scleroderma, lung fibrosis,and cirrhosis), juvenile arthritis, Kawasaki disease, leukemia,lymphoma, lymphoproliferative disorders, multiple sclerosis, myastheniagravis, myeloma, neuromyelitis optica, pemphigus, polymyositis, primarybiliary cholangitis, primary sclerosing cholangitis, psoriasis,psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjögren'ssyndrome, systemic lupus erythematosus, Takayasu's arteritis, temporalarteritis, transplant rejection, transverse myelitis, ulcerativecolitis, uveitis, vasculitis, vitiligo and Vogt-Koyanagi-Harada Disease.

According to an eighth aspect of the invention there is provided anantibody or antigen-binding fragment thereof in accordance with thefirst aspect of the invention or the pharmaceutical composition inaccordance with the sixth aspect of the invention for use in therapy.

In a particular embodiment, the therapy is treatment or prevention of aBTLA-related disease.

In a particular embodiment, the BTLA-related disease is one caused bydecreased expression and/or activity of BTLA in a subject. Inparticular, any disease or disorder characterised by the presence oractivity of T or B cells can be treated with a BTLA agonist antibody orantigen-binding fragment of the invention.

In one embodiment, the BTLA-related disease is an inflammatory disease(such as rheumatoid arthritis), an autoimmune disease or disorder (suchas graft versus host) or a proliferative disease or disorder (such ascancer).

In a particular embodiment, the therapy is treatment or prevention ofinflammatory or autoimmune diseases, and disorders of excessive immunecell proliferation.

According to a variation of the eighth aspect of the invention there isprovided a method of treating a patient in need thereof, comprisingadministering to the patient an antibody or antigen-binding fragmentthereof in accordance with the first aspect of the invention or thepharmaceutical composition in accordance with the sixth aspect of theinvention. In a particular embodiment the patient in need of treatment,or to be treated, has (or is suffering from) a BTLA-related disease. Ina particular embodiment, the patient in need of treatment, or to betreated, has (or is suffering from) an inflammatory disease, anautoimmune disease, or a disorder of excessive immune cellproliferation.

In a particular embodiment, the antibody or antigen-binding fragmentthereof in accordance with the first aspect of the invention or thepharmaceutical composition in accordance with the sixth aspect of theinvention is administered to a patient in need thereof in apharmaceutically acceptable amount.

In a variation of this aspect there is provided an antibody orantigen-binding fragment thereof in accordance with the first aspect ofthe invention or the pharmaceutical composition in accordance with thesixth aspect of the invention for use in a method of treating a patientin need thereof. In a particular embodiment, the method is for treatingor preventing a BTLA-related disease. In particular embodiments, themethod is for treating or preventing inflammatory or autoimmunediseases, and disorders of excessive immune cell proliferation.

In a further variation of this aspect there is provided use of anantibody or antigen-binding fragment thereof in accordance with thefirst aspect of the invention or the pharmaceutical composition inaccordance with the sixth aspect of the invention in the manufacture ofa medicament for the treatment of a patient in need thereof.

In one embodiment, the therapy is for treating a BTLA-related disease.Suitably, the BTLA-related disease is an inflammatory disease (such asasthma), an autoimmune disease or disorder (such as rheumatoidarthritis) or an immunoproliferative disease or disorder (such aslymphoma).

In particular embodiments, the antibody or antigen-binding fragmentthereof of the invention or a pharmaceutical composition comprising saidantibody or antigen-binding fragment thereof is used to suppress T-cellsand/or B-cells.

In particular embodiments, the antibody or antigen-binding fragmentthereof of the invention or a pharmaceutical composition comprising saidantibody or antigen-binding fragment thereof is used for treating orpreventing a disease or condition in a subject in need thereof selectedfrom the group consisting of: Addison's disease, allergy, alopeciaareata, amyotrophic lateral sclerosis, ankylosing spondylitis,anti-phospholipid syndrome, asthma (including allergic asthma),autoimmune haemolytic anaemia, autoimmune hepatitis, autoimmunepancreatitis, autoimmune polyendocrine syndrome, Behcet's disease,bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinatingpolyneuropathy, coeliac disease, Crohn's disease, Cushing's Syndrome,dermatomyositis, diabetes mellitus type 1, eosinophilic granulomatosiswith polyangiitis, graft versus host disease (GVHD), Graves' disease,Guillain-Barre syndrome, Hashimoto's thyroiditis, HidradenitisSuppurativa, inflammatory fibrosis (e.g., scleroderma, lung fibrosis,and cirrhosis), juvenile arthritis, Kawasaki disease, leukemia,lymphoma, lymphoproliferative disorders, multiple sclerosis (MS),myasthenia gravis, myeloma, neuromyelitis optica, pemphigus,polymyositis, primary biliary cholangitis, primary sclerosingcholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis,sarcoidosis, Sjögren's syndrome, systemic lupus erythematosus,Takayasu's arteritis, temporal arteritis, transplant rejection,transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligoand Vogt-Koyanagi-Harada Disease.

In particular embodiments, the antibody or antigen-binding fragmentthereof of the invention or a pharmaceutical composition comprising saidantibody or antigen-binding fragment thereof is used for treating orpreventing a disease or condition in a subject in need thereof selectedfrom the group consisting of: GVHD, colitis, rheumatoid arthritis,psoriasis and MS. In one embodiment, the immunoproliferative disease iscancer. Suitably the cancer is a leukemia or a lymphoma.

In another embodiment, the antibody or antigen-binding fragment thereofof the invention or a pharmaceutical composition comprising saidantibody or antigen-binding fragment thereof is for use in theprevention or treatment of transplant rejection.

In another embodiment, the invention relates to the prevention ortreatment of graft versus host disease.

In another embodiment, the antibody or antigen-binding fragment thereofof the invention or a pharmaceutical composition comprising saidantibody or antigen-binding fragment thereof is for use in the treatmentof rheumatoid arthritis.

In other embodiments, the antibody or antigen-binding fragment thereofof the invention or a pharmaceutical composition comprising saidantibody or antigen-binding fragment thereof is for use in the treatmentof diabetes, such as type 1 diabetes.

In another embodiment, the antibody or antigen-binding fragment thereofof the invention or a pharmaceutical composition comprising saidantibody or antigen-binding fragment thereof is for use in the treatmentof psoriasis.

In another embodiment, the antibody or antigen-binding fragment thereofof the invention or a pharmaceutical composition comprising saidantibody or antigen-binding fragment thereof is for use in the treatmentof multiple sclerosis.

In another embodiment, the antibody or antigen-binding fragment thereofof the invention or a pharmaceutical composition comprising saidantibody or antigen-binding fragment thereof is for use in the treatmentof colitis.

The term “effective amount” or “therapeutically effective amount” refersto a dosage or an amount of a drug that is sufficient to ameliorate thesymptoms in a patient or to achieve a desired biological outcome, e.g.,with cancer, an increased death of tumour cells, reduced tumour size,increased progression free survival or overall survival etc. Asdisclosed elsewhere herein, the effective amount will typically beassessed through extensive human clinical studies.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of them mean “including but notlimited to”, and they are not intended to (and do not) exclude othermoieties, additives, components, integers or steps. Throughout thedescription and claims of this specification, the singular encompassesthe plural unless the context otherwise requires. In particular, wherethe indefinite article is used, the specification is to be understood ascontemplating plurality as well as singularity, unless the contextrequires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith. All of the features disclosed in this specification(including any accompanying claims, abstract and drawings), and/or allof the steps of any method or process so disclosed, may be combined inany combination, except combinations where at least some of suchfeatures and/or steps are mutually exclusive. The invention is notrestricted to the details of any foregoing embodiments. The inventionextends to any novel one, or any novel combination, of the featuresdisclosed in this specification (including any accompanying claims,abstract and drawings), or to any novel one, or any novel combination,of the steps of any method or process so disclosed.

The reader's attention is directed to all papers and documents which arefiled concurrently with or previous to this specification in connectionwith this application and which are open to public inspection with thisspecification, and the contents of all such papers and documents areincorporated herein by reference.

The invention will now be further described with reference to thefollowing non-limiting Examples and accompanying Figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

Binding of antibodies to human and cynomolgus BTLA in soluble and cellexpressed forms. (a) Surface plasmon resonance (SPR) binding curves forsoluble monomeric human BTLA extracellular domain injected at increasingconcentrations over immobilized anti-BTLA antibody; graphs show SPRsignal after reference and blank subtraction. (b) Association anddissociation rates for binding to human or cynomolgus BTLA as calculatedby curve fitting using BiaEvaluation software. (c) Binding of antibody2.8.6, compared to isotype control antibody, to a human BTLA orcynomolgus BTLA expressing Jurkat cell line. (d) EC50s for antibodybinding to transfected cell lines, as calculated by non-linear curvefitting using GraphPad Prism software

FIG. 2

(a) Blockade of ligand binding by anti-BTLA antibodies was assessed bySPR. Human BTLA extracellular domain was immobilized on the sensor chip.Human HVEM was injected to confirm binding, then allowed to fullydissociate. A saturating concentration of anti-BTLA antibody was theninjected, followed immediately by a second injection of HVEM. (b)Equilibrium binding of HVEM after injection of antibody was expressed asa percent of HVEM binding prior to antibody injection. Saturation ofBTLA with clone 11.5.1, but not with clone 2.8.6, blocked subsequentbinding of ligand.

FIG. 3

Epitope mapping of anti-BTLA antibodies. (a) HEK293T cells transfectedwith BTLA constructs in a bicistronic vector also expressing GFP werestained with Pacific Blue conjugated anti-BTLA antibody. Clone 11.5.1binds to cells transfected with wild-type receptor (left) but not tocells transfected with BTLA having a Y39R mutation (right). (b) Bindingto each BTLA mutant construct was expressed as a percentage of bindingto wild-type BTLA for clones 2.8.6 and 11.5.1. (c) Mutations Y39R andK41E which selectively eliminate binding of clone 11.5.1 were mappedonto the crystal structure of human BTLA (black residues). Residuescritical for binding of the ligand HVEM are highlighted in grey.

FIG. 4

(a) The crystal structure of human BTLA extracellular domain in complexwith the Fab′ fragment of clone 2.8.6. Residues on BTLA which are buriedat the interface are highlighted in black. (b) The epitope of antibody2.8.6 is shown (black residues) in relation to the HVEM binding site(grey residues).

FIG. 5

(a) Strategy for creation of a chimeric BTLA gene in humanised-BTLAmice. A section of human genomic DNA from the beginning of exon 2 to theend of exon 3 was inserted into the mouse locus replacing the mousesequence from the beginning of exon 2 to the end of exon 4. Thesequences at the exon-intron junction at the beginning of mouse exon 2and end of mouse exon 4 were left intact to ensure proper splicing.

FIG. 6

(a) Protocol for T cell transfer assay to assess anti-BTLA antibodies invivo. A mixture of humanised and wild-type OVA specific CD4 T cells wasinjected into recipient mice. The next day mice were immunised withovalbumin in Alum to activate the transferred cells and 24 hours laterwere dosed with anti-human-BTLA antibody or isotype control. Eight daysafter initial cell transfer the ratio of humanised to wild-type cells inthe transferred population in the spleen was assessed by flow cytometry.(b) Clone 11.5.1 and to a lesser extent 2.8.6 both reduced expansion ofthe humanised cells relative to the wild-type. Graph shows pooled datafrom two (for 11.5.1) or three (for 2.8.6) repeat experiments.

FIG. 7

Effect of anti-BTLA clone 2.8.6 on CD4 T-cell proliferation in a mixedlymphocyte reaction in vitro. T cells from humanised C57BL/6 mice werestained with CellTraceViolet and added to Mitomycin C treated Balb/cstimulator cells in the presence of anti-BTLA antibody or isotypecontrol. After 96 hours, proliferation of humanised CD4 cells wasassessed and normalised to proliferation in the absence of antibody.Clone 2.8.6 inhibited proliferation of humanised cells with an IC50 of0.029 nM and had a maximal effect of 42% inhibition of proliferation.

FIG. 8

(a) Effect of clone 2.8.6 in a T cell colitis model. RAG knockoutrecipient mice were injected with CD45RBhiCD25-CD4+ T cells fromhumanised BTLA mice and treated with 200 μg 2.8.6 or isotype controlantibody on days 7, 21 and 35. Isotype control treated miceprogressively lost weight from 3 weeks onwards, whilst 2.8.6 treatedmice were spared. (b) 8 weeks after cell transfer colons were processedto extract lamina propria lymphocytes and the total number ofinflammatory cells extracted per colon was calculated. Isotype controltreated mice had significantly more infiltrating immune cells than 2.8.6treated mice. (c) Colon weight to length ratios were calculated as amarker of inflammation and thickening. 2.8.6 treatment prevented theincrease in weight to length ratio seen in isotype control treated mice.

FIG. 9

(a) Effect of BTLA antibodies in a parent-to-F1 model of GVHD. C57BL/6splenocytes and bone marrow cells from humanised-BTLA mice were injectedinto CB6F1 recipient mice, which were then treated with anti-BTLAantibody or isotype control. Untreated mice developed clinical GVHD withprogressive weight loss, dermatitis and diarrhea and were culled whenthey reached pre-specified humane endpoints. 2.8.6 and 11.5.1 antibodytreated mice were relatively spared, with survival comparable to controlmice reconstituted with syngeneic cells. (b) 5 weeks after cell transfermice were culled and colon weight to length ratio was calculated as amarker of gut inflammation. 2.8.6 and 11.5.1 treatment prevented thecolon thickening seen in untreated mice.

FIG. 10

(a) Effect of D265A mutated clone 11.5.1 in a T cell transfer assay invivo. This mutated antibody, which does not bind Fc receptors, no longerinhibited proliferation of humanised BTLA cells, instead lead toenhanced proliferation due to receptor blockade. (b) The D265A mutated11.5.1 antibody no longer inhibited T cell proliferation in a mixedlymphocyte reaction.

FIG. 11

Anti-BTLA antibodies do not fix complement. Splenocytes from humanisedBTLA mice were incubated with 10% rabbit complement for 1 hour at 37° C.in the presence of 20 μg/ml BTLA antibody, isotype control or positivecontrol (a depleting CD20 antibody). Anti-CD20 antibody depleted themajority of B cells confirming the activity of the rabbit complement,but BTLA antibodies did not deplete either B or T cells, even thoughboth these populations stain positive for BTLA.

FIG. 12

Anti-BTLA antibodies do not cause antibody-dependent-cell-mediatedcytotoxicity. Splenocytes from humanised BTLA mice were incubated for 24hours at 37° C. in the presence of 20 μg/ml BTLA antibody, isotypecontrol or positive control (a depleting CD20 antibody). Anti-CD20antibody depleted the majority of B cells by inducing ADCC by effectorcells in the mixture, but BTLA antibodies did not deplete either B or Tcells, even though both these populations stain positive for BTLA.

FIG. 13

Anti-BTLA antibodies do not deplete B or T cells in vivo. Humanised BTLAmice were injected with 200 μg of 2.8.6 antibody. At 24 hours spleensand bone marrow were collected and cell populations assessed by flowcytometry. 2.8.6 did not deplete B or T cells in the spleen or affectthe frequency of different B cell precursor populations in the bonemarrow.

FIG. 14

BTLA expression levels on B cells or CD4⁺ T cells from humanised micefollowing 6 days of in vivo incubation with antibodies 2.8.6 or 11.5.1,compared to BTLA expression on cells from mice injected with isotypecontrol antibody.

EXAMPLES

In the examples that follow it is shown that antibodies 11.5.1 and 2.8.6bind to human BTLA with high affinity. Using transgenic mice expressingthe human receptor it is shown that, following binding to BTLA, theseantibodies inhibit T cell responses in vitro and in vivo and are able toameliorate disease in murine models of inflammatory bowel disease andgraft-versus-host disease. Whilst these agonist effects are dependent onFc-receptor binding, the antibodies do not cause depletion of BTLAexpressing cells via cytotoxicity and do not induce receptordown-modulation.

Example 1 Generation and Sequencing of Anti-BTLA Antibodies

Antibodies recognizing the human immune cell receptor BTLA weregenerated by BioGenes GmbH via immunizing mice with the extracellularregion of human BTLA (BTLA^(K31-R151)). Splenocytes from immunized micewere fused with Sp2/0-Ag14 myeloma cells and resulting hybridomasselected for reactivity with human BTLA by ELISA of supernatants, inconjunction with dilution cloning. Antibodies were isotyped fromhybridoma supernatant using a Rapid Mouse Isotyping Kit (RayBiotech).The antibodies produced by clones 2.8.6 and 11.5.1 were both found to beIgGlk.

To sequence the immunoglobulin variable domains, RNA was extracted fromhybridomas using TRIzol Reagent (ThermoFisher) as per the manufacturer'sinstructions. RNA was reverse transcribed to produce cDNA using primersspecific for the first constant domain of the heavy chain or for theconstant domain of the light chain, and Super Script II ReverseTranscriptase (Invitrogen) as per manufacturer's instructions.

PCR was then performed using primers targeting conserved regions of theimmunoglobulin locus as previously described (Tiller et al., J ImmunolMethods. 350:183-193, 2009) and PCR products were sequenced. In somecases identification of functional light chain was complicated byabundant non-functional kappa light chain cDNA from the fusion myelomacell line, and to resolve this a previously described technique wasemployed, adding excess primer specific for the non-functional chainCDR3 to force truncation of the aberrant chain product (Yuan et al. JImmunol Methods. 294:39553-61, 2005).

Variable domain sequences were assessed using the NCBI IgBlast tool todetermine the location of the CDRs.

Example 2 Binding to Soluble Human and Cynomolgus BTLA

The binding affinity and kinetics of the BTLA agonist antibodies of thepresent invention (2.8.6 and 11.5.1) to human or cynomolgus BTLA weredetermined by surface plasmon resonance using the Biacore T200 (GEHealthcare). Mouse antibody capture kit (GE Healthcare) was used to coata Series S CM5 Sensor Chip (GE Healthcare) with polyclonal anti-mouseIgG. Anti-BTLA antibody was then captured onto the biosensor surface anda negative control antibody (clone Mopc21; Biolegend) captured in thereference channel. Various concentrations of monomeric soluble humanBTLA extracellular domain (BTLA^(K31-R151)) (from SEQ ID NO: 23) orsoluble cynomolgus macaque BTLA extracellular domain (BTLA^(K31-R151))(from SEQ ID NO: 24) were then injected over the immobilized antibodiesin the buffer 10 mM Hepes, 150 mM NaCl, 0.005% v/v Surfactant P20, pH7.4 (HBS-P) at 37° C., in a single cycle kinetics analysis (FIG. 1a ).Association and dissociation rates were fitted using BiaEvaluationSoftware (GE Healthcare) after reference and blank subtractions, anddissociation constants were calculated (FIG. 1b ). Clone 2.8.6 bindshuman BTLA with a KD of 0.65 nM and cynomolgus BTLA with a KD of 7.89nM. Clone 11.5.1 binds human BTLA with a KD of 0.75 nM and cynomolgusBTLA with a KD of 0.99 nM. In a separate experiment against human BTLAonly, Clone 2.8.6 bound human BTLA with a KD of 0.37 nM and Clone 11.5.1boundds human BTLA with a KD of 0.53 nM.

Example 3 Binding to BTLA on Cells

The ability of the BTLA agonist antibodies of the present invention(2.8.6 and 11.5.1) to bind to human or cynomolgus BTLA expressed on thecell surface was assessed by flow cytometry. A lentiviral transfectionsystem was used to express full length human or cynomolgus BTLA in aJurkat T cell line. 1×10⁵ cells per well were plated in 96 well U-bottomplates. BTLA antibody binding versus mIgG1 isotype control (cloneMOPC-21, Biolegend #400165) was assessed at twelve concentrations by 1in 3 serial dilution in FACS buffer (PBS, 2% FCS, 0.05% sodium azide),starting at a concentration of 90 μg/ml. Non-specific antibody bindingwas prevented by addition of Fc block (Biolegend #101319). Antibodieswere incubated with cells for 30 minutes on ice, then cells were washedtwice with FACS buffer prior to staining with an AF647 conjugatedanti-mIgG1 secondary antibody (Biolegend #406618). Secondary antibodywas incubated for 30 minutes on ice, then cells were washed andresuspended in FACS buffer for analysis on a flow cytometer. Thegeometric mean fluorescent intensity of secondary antibody was plottedfor each concentration and the EC50 for receptor binding calculated bynon-linear curve fitting using GraphPad Prism software. Clone 11.5.1binds to human BTLA expressing cells with an EC50 of 0.016 nM andcynomolgus BTLA expressing cells with an EC50 of 0.0057 nM. Clone 2.8.6binds to human BTLA expressing cells with an EC50 of 0.085 nM andcynomolgus BTLA expressing cells with an EC50 of 0.16 nM (FIG. 1c-d ).

Example 4 Competition with the Natural Ligand HVEM for Binding to BTLA

The ability of the BTLA agonist antibodies of the present invention(2.8.6 and 11.5.1) to block natural ligand binding to BTLA was assessedby surface plasmon resonance using the Biacore T200 (GE Healthcare).Human BTLA extracellular domain (BTLA^(31K-151R)) was covalently coupledto a CMS Sensor chip using amine coupling. Human HVEM extracellulardomain, fused to mouse IgG1 Fc, was then injected over the immobilizedhBTLA in HBS-P buffer at 37° C., and allowed to fully dissociate. Asaturating amount of anti-BTLA antibody (2.8.6 or 11.5.1) was theninjected, followed immediately by a second injection of human HVEM-mFcat the same concentration as the initial injection (FIG. 2a ).Equilibrium HVEM binding (in Resonance Units) after saturation of BTLAwith antibody was expressed as a percentage of binding prior to antibodyinjection (FIG. 2b ). If HVEM binding following saturation with antibodywas >90% of the binding prior to antibody injection then the antibodywas considered non-blocking.

Example 5 Binding Epitope of Antibody 11.5.1 on Human BTLA

The functional epitope of the antibody 11.5.1 on human BTLA wasdetermined by flow cytometry assessment of binding to a panel of singleresidue mutants of the receptor expressed on the cell surface.Constructs encoding the human extracellular region of BTLA with thetransmembrane and intracellular regions of murine CD28 were cloned intothe bi-cistronic mammalian expression vector pGFP2-n2 (BioSignal PackardLtd), which also encodes GFP. Mutant constructs varying by one aminoacid were prepared using the “drastic” mutagenesis approach (Davis etal. Proc Natl Acad Sci USA. 95, 5490-4 (1998)). Plasmids (2 μg/well)were transfected into HEK-293T cells in 6 well plates using Genejuicetransfection reagent (Novagen; 6 μl/well). Mock and no-transfectioncontrols were included with each experiment. Cells were harvested at 48hours and stained with fluorochrome-conjugated anti-BTLA antibody at 10μg/ml, alongside a Live/Dead marker, in PBS, 0.05% azide, 2% FCS (FACSbuffer) for 1 h at 4° C. Cells were washed, pelleted and resuspended in200 μl FACS buffer before being analysed on a BD FACSCanto flowcytometer. GFP-positive (transfected) viable cells were gated andanalysed for binding of anti-BTLA antibodies (an example of the bindinganalysis for clone 11.5.1 is shown in FIG. 3a ). For each mutant theGeo-mean of anti-BTLA antibody binding to transfected cells wasexpressed as a percentage of binding to the wild-type receptor (FIG. 3b). A panel of anti-BTLA antibodies was assessed and any mutation thateliminated binding of all antibodies was excluded from the analysis, onthe assumption that such mutations lead to drastic changes in proteinfolding or expression rather than indicating an antibody epitope. Themutations Y39R and K41E completely abolish binding of antibody 11.5.1whilst leaving binding of 2.8.6 unaffected. These mutations are mappedonto the human BTLA crystal structure (Compaan et al., J Biol Chem.280:39553-61, 2005) in FIG. 3c (black residues), indicating the bindingepitope of 11.5.1. Residues required for HVEM binding (Gln37, Arg42,Pro59, His127; from patent publication number WO2017004213) are alsomapped onto the structure in grey demonstrating that 11.5.1 binds to anepitope very close to the HVEM binding site.

Example 6. Crystal structure of the Fab′ fragment of 2.8.6 in complexwith human BTLA The structural epitope of antibody 2.8.6 on human BTLAwas determined by solving the crystal structure of antibody Fab incomplex with human BTLA extracellular domain. The heavy and lightvariable domains of antibody 2.8.6 were cloned into the pOPINVH andpOPINVL expression vectors (Addgene), which encode the first constantdomain of the mouse IgG1 heavy chain (with a 6xHistidine tag) and theconstant domain of the mouse Ig kappa chain, respectively. These vectorswere transiently co-transfected into HEK293T cells to produce the Fab′fragment of anti-BTLA 2.8.6, which was purified by Ni-NTA purification.Human BTLA Ig-V set domain (BTLA^(S33-D135)) was cloned into the pGMT7vector and expressed in BL21(DE3)pLysS E. coli cells (Novagen) toproduce inclusion bodies. The inclusion bodies were isolated from thecell pellet by sonication and washed repeatedly with a wash solutioncontaining 0.5% Triton X-100. The purified BTLA inclusion bodies weresolubilized in a denaturant solution containing 6 M guanidinehydrochloride. The solubilized protein solution was diluted slowly inrefolding buffer [0.1 M Tris-HCl (pH 8.0), 0.6 M L-arginine, 2 mMethylenediaminetetraacetic acid, 3.73 mM cystamine, and 6.73 mMcysteamine] to a final protein concentration of 1-2 μM and then stirredfor 48 h at 4° C. The refolded mixture of BTLA was then concentratedwith a VIVA FLOW50 system (Sartorius). BTLA was purified by gelfiltration on a Superdex 75 column (GE Healthcare).

The purified BTLA and Fab′ were mixed and purified as a complex by sizeexclusion chromatography. The crystal suitable for data collection wasobtained in 0.2 M calcium acetate, 0.1 M imidazole pH 8.0, 10% (w/v) PEG8000 at 293° K by the hanging drop vapor-diffusion method. The finaldataset was collected at the Photon Factory, and the structure wasdetermined by molecular replacement using the structure of BTLA (PDB ID;2AW2 chain A) and anti-PD1-Fab (PDB ID: SGGS chain C, D) as searchprobes.

The residues on BTLA at the interface with antibody 2.8.6 are A50, G51,D52, P53, E83, D84, R85, Q86, E103, P104, V105, L106, P107, N108, D135.

Example 7 Development of Humanised BTLA Mice

To provide a platform to assess anti human-BTLA antibodies in mousemodels, a knock-in strain of C57Bl/6 mice was developed expressing achimeric form of BTLA with the human extracellular region and the murinetransmembrane and signaling regions. A section of human genomic DNA fromthe beginning of exon 2 to the end of exon 3 was inserted into the mouselocus replacing the mouse sequence from the beginning of exon 2 to theend of exon 4. The sequences at the exon-intron junction at thebeginning of mouse exon 2 and end of mouse exon 4 were left intact toensure proper splicing (FIG. 5).

Example 8 Inhibition of Antigen-Specific T Cell Proliferation In Vivo

The ability of the BTLA agonist antibodies of the present invention(2.8.6 and 11.5.1) to inhibit antigen specific T cell proliferation invivo was assessed using a sensitive T-cell transfer assay (FIG. 6a ). Inthis assay, 5×10⁵ T-cells, comprising a mixture of purified OTII (TCRtransgenic) CD4⁺ T cells specific for ovalbumin (OVA) from miceexpressing homozygous human BTLA (hBTLA), and from OT-II mice expressingthe wild-type murine BTLA receptor (The Jackson Laboratory), weretransferred into non-transgenic C57BL/6 recipients. The transferredcells were distinguished from host cells using the CD45.2 (versusCD45.1) allotypic marker. The wild-type donor cells also expressed greenfluorescent protein under the control of the human ubiquitin C promoterto allow them to be distinguished from the humanised donor cells by flowcytometry. The day after T cell transfer, the recipient mice wereimmunised with 100 μg ovalbumin (Sigma-Aldrich) in 100 μl PBS mixed with100 μl Imject Alum (ThermoFisher), to induce expansion of the T cells.On the second day, the mice were dosed with 200 μg of antibody,intraperitoneally. Eight days following the initial transfer of the Tcells, the ratio of the humanised BTLA-expressing and wild-typeOVA-specific T-cells in the spleen was determined by flow cytometry. Inthis way, it was possible to track the expansion or contraction of thehumanised cells, which bind the anti-human BTLA antibodies, relative tothe wild-type controls, which do not. Both antibodies 2.8.6 and 11.5.1led to reduced expansion of the humanised BTLA cells relative to thewild-type controls indicating that they are inducing signaling throughthe inhibitory BTLA receptor, which leads to reduced T cellproliferation (FIG. 6b ).

Example 9 Inhibition of T Cell Proliferation in a Mixed LymphocyteReaction

The ability of the BTLA agonist antibodies of the present invention(2.8.6 and 11.5.1) to inhibit proliferation of primary T cells from thehumanised mice in vitro was assessed using a mixed lymphocyte reaction(MLR). Splenocytes from Balb/c mice were treated with Mitomycin C for 30mins at 37° C. then washed and used as stimulator cells. T cells werepurified from the spleens of humanised BTLA mice, by negative selectionusing magnetic-activated cell sorting (Mojosort Mouse CD3 T cellisolation kit, Biolegend #480023), and stained with CellTrace VioletCell Proliferation Kit (ThermoFisher) to use as responder cells. 4×10⁵stimulator cells and 2×10⁵ responder cells per well were mixed in96-well U-bottom plates with various concentrations of anti-BTLA orisotype control antibody (clone MOPC-21, Biolegend #400165). Serial 1 in3 dilutions of antibody were assessed starting at a concentration of1μg/ml for a total of 10 concentrations. Polyclonal anti-mHVEM antibody(R&D systems #AF2516) was also added to all wells at 1411 μg/ml to blockany baseline signaling through the BTLA pathway and accentuate theeffects of agonist antibodies. After 96 hours, dilution of CellTraceViolet in responder cells was assessed by flow cytometry as a marker ofproliferation. Proliferation in the presence of anti-BTLA antibody orisotype control was compared to proliferation in the absence ofantibody. CD4⁺ and CD8⁺ populations were gated out and analysedseparately. Both antibodies 2.8.6 and 11.5.1 reduced proliferation ofhuman-BTLA expressing T cells, indicating that they induce inhibitorysignaling through the human BTLA receptor. Clone 2.8.6 inhibited CD4 Tcells with an IC50 of 0.029 nM and had a maximal effect of 42%inhibition of proliferation (FIG. 7). Clone 11.5.1 inhibited CD4 T cellswith an IC50 of 0.016 nM and had a maximal effect of 33% inhibition ofproliferation.

Example 10 Inhibition of NFkB Signalling in Human BTLA or CynomolgusBTLA Transfected Jurkat T Cell Lines

The ability of the BTLA agonist antibodies of the present invention(2.8.6 and 11.5.1) to inhibit NFkB signalling was assessed using a BTLAtransfected reporter T cell line. A Jurkat T cell line stablytransfected with an expression cassette that includes NF-κB-responsivetranscriptional elements upstream of a minimal CMV promoter (mCMV)-GFPcassette (Source BioSciences #TR850A-1) was used as a reporter cell linefor NFkB signalling. A lentiviral transfection system was used toexpress full length human or cynomolgus BTLA in this reporter cell line.These cells were mixed with a stimulator cell line comprised of bw5147cells expressing an anti-CD3 ScFv construct on their surface asdescribed by Leitner et al. J Immunol Methods. 2010 Oct. 31;362(1-2):131-41. The stimulator cell line was also transfected withmurine FcyRIIB to provide Fc receptors for presentation of the agonistBTLA antibodies. 5×10⁴ reporter cells per well were mixed in 96 wellU-bottom plates with 5×10⁴ stimulator cells in the presence of variousconcentrations of BTLA antibody or isotype control (clone MOPC-21,Biolegend #400165). After 24 hours incubation at 37° C., cells werepelleted and stained for flow cytometry with a viability dye (ZombieAqua, Biolegend #423101) and a mouse CD45 antibody (Pe-Cy7 conjugatedclone 104, Biolegend #109830) to separate stimulator (murine) fromresponder (human) cells. Geometric mean of GFP expression was assessedfor each antibody concentration and normalized to GFP expression in theabsence of antibody. Clone 2.8.6 inhibited human BTLA transfected cellswith an IC50 of 0.06 nM and cynomolgus BTLA transfected cells with anIC50 of 0.22 nM. Clone 11.5.1 inhibited human BTLA transfected cellswith an IC50 of 0.033 nM and cynomolgus BTLA transfected cells with anIC50 of 0.14 nM.

Example 11 Treatment of a T Cell Driven Mouse Model of Colitis byAntibody 2.8.6

The ability of the BTLA agonist antibody 2.8.6 to ameliorate a T celldriven model of colitis was assessed using the humanised mice. This Tcell transfer model has previously been described as a murine model ofinflammatory bowel disease (Ostanin et al., Am J Physiol GastrointestLiver Physiol. 296:G135-46, 2009). CD45RB^(hi)CD25-CD4+ T cells sortedfrom spleens and lymph nodes of humanised BTLA mice were injectedintraperitoneally into Rag1 KO recipients, (Rag1^(tm1Mom); The JacksonLaboratory), at a dose of 5×10⁵ cells per mouse. The transferred T cellscause an inflammatory colitis that develops after approximately 3 weeksand leads to diarrhea and weight loss. Rag1 KO cagemates that did notreceive transferred T cells serve as non-diseased controls. On days 7,21 and 35 after T cell transfer the recipient mice were injectedintraperitoneally with 200 μg of 2.8.6 or isotype control antibody. Allmice were weighed regularly, and at 8 weeks colons were weighed andmeasured and inflammatory infiltration assessed by histology, as well asby cell counting and flow cytometry of extracted lamina proprialeucocytes. Antibody 2.8.6 prevented weight loss (FIG. 8a ) andsignificantly reduced inflammatory infiltration of colons (FIG. 8b ).Colon inflammation in diseased mice led to an increased colonweight:length ratio that was not seen in 2.8.6 treated mice (FIG. 8c ).

Example 12 Treatment of a Mouse Model of Graft-Versus-Host Disease(GVHD)

The effects of the anti-BTLA agonist antibodies were assessed in anon-lethal parent-into-F1 model of GVHD. Bone marrow cells (BMCs) andsplenocytes were harvested from humanised BTLA donor mice (C57BL/6background; H2^(b)). 2×10⁷ BMCs and 107 splenocytes were injectedintravenously into CB6F1 (H2^(b/d)) recipients that had been lethallyirradiated with 9 Gy total body irradiation. Irradiated CB6F1 micereconstituted with syngeneic BMCs and splenocytes served as non-diseasedcontrols. On the day of immune cell transfer mice were injectedintraperitoneally with 200 μg anti-BTLA antibody or isotype control.Mice were weighed regularly and GVHD was monitored by calculatingrelative loss of body weight and by clinical observation. Mice wereculled 5 weeks after immune cell transfer or when they reached a humaneendpoint (which included >20% weight loss relative to starting weight inthe first 14 days, or >15% weight loss at any other time). At the timeof death colons were weighed and measured and a colon weight:lengthratio calculated as a marker of colon inflammation, which is a prominentclinical feature of GVHD. Both antibodies 2.8.6 and 11.5.1 significantlyreduced weight loss, leading to increased survival (FIG. 9a ) andprevented colon inflammation (FIG. 9b ).

Example 13 Agonist Activity of Antibody 11.5.1 is Dependent on FcReceptor Binding

Antibody 11.5.1 was recombinantly expressed as a mIgGlk containing aD265A mutation which has previously been described as significantlyreducing Fc receptor binding (Clynes et al., Nat Med. 6:443-446, 2000).This mutated antibody was assessed in the T cell transfer assaydescribed in Example 8. The parental 11.5.1 antibody inhibitedproliferation of humanised T cells as its net effect is agonism of theBTLA receptor. The FcR-null D265A mutation, however, led to enhancedproliferation of humanised T cells suggesting that the FcR-null mutationremoves the antibody's agonistic effect, leaving only the effect ofreceptor blockade (FIG. 10a ).

The D265A mutated 11.5.1 antibody was also assessed in the in vitro MLRassay described in Example 9. Again, the parental 11.5.1 antibodyinhibited proliferation of humanised T cells as its net effect isagonism of the BTLA receptor. The FcR-null D265A mutation removes theantibody's agonistic effect, so this antibody showed no effect in thisassay (FIG. 10b ). The FcR null 11.5.1 antibody did not enhanceproliferation of humanised cells in this assay as HVEM was blocked (bythe addition of polyclonal anti-HVEM antibody) so there was no baselinesignaling through the pathway to be blocked by the BTLA blockingantibody.

Example 14 Antibodies 2.8.6 and 11.5.1 Do Not Fix Complement In Vitro

Splenocytes from humanised mice were incubated with 10% baby rabbitcomplement (BioRad) and anti-BTLA antibodies (or an isotype control or apositive control depleting anti-CD20 antibody; clone SA271G2 fromBiolegend) at 20 μg/ml for 15 min at 37° C. Whilst anti-CD20 antibodydepleted the majority of B220⁺ B cells, anti-BTLA antibodies did notdeplete either B220⁺ or CD4⁺ cells (FIG. 11), even though both thesepopulations stain positively for BTLA.

Example 15 Antibodies 2.8.6 and 11.5.1 Do Not Induce ADCC In Vitro

Whole splenocytes (including myeloid effector cells) from humanised micewere incubated with anti-BTLA antibodies (or isotype control ordepleting anti-CD20 antibody SA271G2) at 20 μg/ml for 24 hours at 37° C.Whilst anti-CD20 antibody depleted the majority of B220⁺ cells,anti-BTLA antibodies did not deplete either B220⁺ or CD4⁺ cells (FIG.12), even though both these populations stain positively for BTLA.

Example 16 Antibodies 2.8.6 and 11.5.1 Do Not Deplete BTLA ExpressingCells In Vivo

Humanised BTLA mice were injected intraperitoneally with 200 μganti-BTLA antibody or isotype control. At 24 hours spleens wereharvested and the frequency of different cell populations identified byflow cytometry. Anti-BTLA antibody had no effect on the frequency orabsolute number of B or T cells in the spleen or on the number of B cellprecursors in the bone marrow (FIG. 13).

Example 17 Antibodies 2.8.6 and 11.5.1 Stabilize Expression of BTLA onImmune Cells In Vivo

Humanised mice were injected intraperitoneally with 10 mg/kg of antibody2.8.6 or 11.5.1. Six days after injection mice were humanely sacrificedand spleens harvested and processed to single cell suspension forassessment by flow cytometry. Cells were stained with a cocktail ofantibodies to identify immune cell subsets and with fluorescentlyconjugated anti-BTLA antibody that had a non-competing epitope with theantibody that had been injected. The geometric mean of BTLA stainingfollowing in vivo incubation with anti-BTLA antibody was normalized tothe geometric mean of BTLA staining (using the same staining antibody)following incubation with isotype control. BTLA expression wassignificantly higher on B cells and CD4 T cells from mice that had beeninjected with either clone 2.8.6 or 11.5.1, compared to mice that hadbeen injected with isotype control (FIG. 14). This suggests that clones2.8.6 and 11.5.1 stabilise expression of BTLA on the cell surface invivo, rather than inducing receptor down-modulation, as has beenobserved with other BTLA antibodies in the prior art (M.-L. del Rio etal./Immunobiology 215 (2010) 570-578). For the purposes ofimmunosuppression an agonist antibody that stabilizes expression of thereceptor presents the benefit of enabling prolonged high levels ofinhibitory signaling through the pathway compared to a downmodulatingantibody.

Example 18 Tolerability and Side Effects in Animal Models

There were no tolerability issues or side effects noted in any animalstudies with antibodies 2.8.6 or 11.5.1.

Example 19 Humanisation of Antibody 2.8.6

Antibody 2.8.6 was humanised by CDR grafting on to homologous humangermline framework regions (See SEQ ID NO: 13 -14). IGHV2-5*08 was usedfor the heavy chain and IGKV3-11*01 for the light chain. Afterhumanisation, binding to BTLA was assessed by SPR. Humanised 2.8.6 boundto monomeric BTLA with a K_(D) of 0.73 nM.

Example 20 Characterisation of Exemplary BTLA Antibodies

Described in this example is characterisation of exemplary mIgG1 BTLAantibodies provided herein in addition to 2.8.6 and 11.5.1. Variousclones listed in Table 1 were evaluated for their binding affinity toBTLA and inhibition efficiency of lymphocytes (Table 2). For eachantibody, the association rate (“on rate”) and dissociation rate (“offrate”) for binding human BTLA, and KD for binding human or cynomolgusBTLA were measured according to the method described in Example 2,fitting curves for injection of BTLA extracellular domain at a singleconcentration. Inhibition efficiency of individual antibodies on T cellswas also evaluated at a single concentration of 10 μg/ml. MLR assay wasperformed for each individual antibody according to the method asdescribed in Example 9 (two biological repeats as shown in Table 3);anti-CD3 assay was performed according to the method described below(two biological repeats, Table 3); and inhibition of NFkB signalling inhuman BTLA transfected Jurkat T cell line by each antibody wasdetermined according to the method as described in Example 10 (Table 3).The average inhibition of T cells relative to isotype control in variousin vitro stimulation assays for each exemplary antibody was calculatedas a mean of the percentage inhibition of all assay results (Table 2 andTable 3).

The ability of the BTLA agonist antibodies to inhibit anti-CD3 andanti-CD28 induced T cell activation was assessed as follows. Splenocytesfrom humanised BTLA mice were processed to single cell suspension andtreated with ACK buffer to lyse red blood cells. Cells were stained withCFSE (Biolegend Cat #423801) to enable tracking of cell proliferation.2×10⁵ cells per well were plated in 96 well U-bottom plates with solubleanti-CD3 antibody (clone 145.2C11; Biolegend #100339) and anti-CD28(clone 37.51; Biolegend #102115) each at a concentration of 50 ng/ml,and soluble anti-BTLA antibody or isotype control at a concentration of10 μg/ml. After 72 hours cells were analysed by flow cytometry to assessproliferation (“antiCD3/CD28 (CD4 T cell proliferation)”) and T cellactivation by staining of surface expressed activation markers(“antiCD3/CD28 (CD69+ CD4 T cells)”). For each BTLA antibody thepercentage inhibition compared to isotype control antibody wascalculated.

Further, for each BTLA antibody, their ligand blocking capability, e.g.,competition with HVEM for binding to BTLA, was assessed according to themethod as described in Example 4, and the results are presented as “Yes”for more than 90% inhibition of HVEM-BTLA binding, and “No” for lessthan 10% inhibition of HVEM-BTLA binding. Functional epitope of eachBTLA antibody was also determined according to the method as describedin Example 5. The “epitope” column in Table 2 summarizes the epitopegroup that each individual BTLA antibody binds to. Antibodies 2.8.6,6.2, 831, 16H2, 7A1, 16F10, 6G8, 3E8, 4E8, 15C6, 12F11, 10B1, 15B6, 4D3,16E1, 4D5 and 3A9 all bind to a first epitope (named “epitope 1” in thetable) comprising at least one critical residue selected from the list:D52, P53, E55, E57, E83, Q86, E103, L106 and E92. Antibodies binding toepitope 1 do not compete with the ligand HVEM for binding to BTLA.Antibodies 11.5.1, 14D4, 1H6, 8C4, 27G9, 26F3 all bind to a differentsecond epitope (“epitope 2”) comprising at least one critical residueselected from the list: Y39, K41, R42, Q43, E45 and S47. Antibodiesbinding to epitope 2 do compete with the ligand HVEM for binding toBTLA. Antibody 26B1 binds to a third epitope (“epitope 3”) comprising atleast one critical residue selected from the list: D35, T78, K81, S121and L123. Antibodies binding to epitope 3 do compete with the ligandHVEM for binding to BTLA. Antibodies 24H7, 4B1, 8B4, 4H4 all bind to adifferent fourth epitope (“epitope 4”) comprising the critical residueH68. Antibodies binding to epitope 4 do not compete with the ligand HVEMfor binding to BTLA. Antibody 21C7 binds to a different fifth epitope(“epitope 5”) comprising at least one critical residue selected from thelist: N65 and A64. Antibodies binding to epitope 5 do not compete withthe ligand HVEM for binding to BTLA.

Example 21 Humanisation and CDR Engineering of BTLA Antibodies 3E8 and6.2

The variable domains of 3E8 and 6.2 were humanised by germlining tohomologous human germline framework regions (Seq ID No. 382-385). For3E8 the acceptor frameworks selected were VH1-1-08 and JH6 for the heavychain and VK3-L6 and JK2 for the light chain. For 6.2 the acceptorframeworks selected were VH3-3-21 and JH6 for the heavy chain andVK2-A19 and JK4 for the light chain.

It is sometimes possible to substitute certain residues in the CDRs ofan antibody to remove undesirable characteristics without significantlyimpacting target binding. The CDRH2 of the humanised antibody 6.2 wasmodified with D54E and N56Q substitutions (Seq ID No. 387) to removedeamidation potential (the engineered humanised VH sequence of 6.2 isgiven in Seq ID No. 390). Similarly, the CDRH2 of the humanised antibody3E8 was modified with an N57Q substitution (Seq ID No. 388) to removedeamidation potential, and a K63S substitution to reduce predictedimmunogenicity, as determined by Lonza's Epibase analysis (theengineered humanised VH sequence of 3E8 is given in Seq ID No. 389).

TABLE 2 Characterisation of binding affinity and inhibitory effect ofexemplary antibodies Human Human Human BTLA On BTLA BTLA Cyno AverageLigand rate Off rate KD BTLA KD inhibitory Clone Blocking (1/Ms) (1/s)(nM) (nM) effect in vitro Epitope 2.8.6 No 6.46E+05 4.23E−04 0.65 7.8939% 1 24H7 No 2.43E+05 1.60E−04 0.66 — 30% 4 11.5.1 Yes 6.03E+054.49E−04 0.75 0.99 30% 2 14D4 Yes 2.54E+05 3.77E−04 1.49 1.83 33% 2 6.2No 6.30E+05 1.07E−03 1.70 9.71 35% 1 4B1 No 5.77E+05 1.85E−03 3.21 — 29%4 8B4 No 5.38E+05 4.40E−03 8.17 — 29% 4 16H2 No 3.97E+05 3.27E−03 8.25160.1 34% 1 1H6 Yes 7.72E+05 6.90E−03 8.94 6.08 31% 2 8C4 Yes 3.63E+055.76E−03 15.89 161.48 19% 2 26B1 Yes 3.23E+05 9.70E−03 30.03 167.66 21%3 7A1 No 4.13E+05 1.66E−02 40.17 — 24% 1 21C7 No 9.30E+05 4.06E−02 43.65— 18% 5 16F10 No 5.81E+05 2.83E−02 48.78 — — 1 6G8 No 3.18E+05 1.67E−0252.42 — — 1 3E8 No 5.43E+05 6.08E−02 111.98 607.46 41% 1 4E8 No 1.75E+053.14E−02 180.00 — — 1 27G9 Yes 1.92E+05 8.38E−02 436.86 653.63 16% 215C6 No 1.93E+05 1.38E−01 718.44 — — 1 12F11 No 2.15E+05 1.55E−01 722.33— 24% 1 10B1 No 4.22E+05 5.21E−01 1233.36 — 21% 1 15B6 No 4.47E+055.76E−01 1287.18 — 14% 1 4D3 No 1.52E+05 2.51E−01 1651.32 — — 1 4H4 No2.03E+05 3.47E−01 1708.23 — 26% 4 26F3 Yes 9.21E+05 2.02E+00 2195.81809.75  9% 2 16E1 No 7.30E+05 2.13E+00 2923.69 — 15% 1 4D5 No 2.70E+057.90E−01 2929.18 — — 1 3A9 No 4.06E+05 1.63E+00 4006.90 — 19% 1

TABLE 3 Inhibitory effect assay results of exemplary antibodiesAntiCD3/CD28 AntiCD3/CD28 T cell MLR (CD4 T cell (CD4 T cell (CD69 + CD4T reporter proliferation) proliferation) cells) (NFκB Clone repeat 1repeat 2 repeat 1 repeat 2 repeat 1 repeat 2 signaling) Average 2.8.630% 36% 23% 35% 58% 67% 22% 39% 24H7 23% 31% 13% 23% 52% 44% 22% 30% 6.231% 35% 19% 21% 53% 61% 26% 35% 11.5.1 23% 18% 21% 28% 50% 47% 19% 30%11.5.1 D265A −3%  1% −3% −9% −47%  −26%  −13%  −14%  4B1 33% 30% 14% 18%47% 41% 23% 29% 14D4 39% 26% 24% 29% 43% 52% 16% 33% 831 25% 34% 10%  8%50% 53% 24% 29% 16H2 40% 26% 11% 23% 51% 60% 29% 34% 1H6 31% 16% 26% 19%47% 53% 26% 31% 8B4 33% 23% 20%  4% 51% 47% 24% 29% 21C7  8% 17% 10% −4%39% 35% 23% 18% 3E8 43% 35% 27% 35% 52% 64% 30% 41% 7A1 23% 29% 14% 17%28% 38% 20% 24% 26B1 12% 10% 11% 19% 35% 30% 29% 21% 8C4 42% −2% 12%  4%29% 29% 21% 19% 27G9  9%  8% 10% 13% 24% 22% 24% 16% 12F11 28% 23%  5% 9% 30% 40% 30% 24% 15C6 19%  8%  2% −2% 12% 19%  9% 10% 26F3  9% −5% 4%  0% 19% 17% 20%  9% 4D3 12%  9% −4% −2%  6%  2% 26%  7% 10B1 16% 25% 8% 14% 24% 36% 27% 21% 16E1 33%  8%  4%  8%  9% 23% 22% 15% 15B6  7%13%  9% 16% 13% 20% 21% 14% 3A9  7% 24%  9%  9% 22% 34% 27% 19% 4H4 10%17% 14% 22% 43% 52% 25% 26% No antibody  3% −3%  1% −6%  2% −9%  2% −1%

SEQUENCE LISTING: SEQ ID NO: 1 GDSITSAY SEQ ID NO: 2 ISYSGSTSEQ ID NO: 3 ARSHYYGYYFDY SEQ ID NO: 4 ETIDSYGDSL SEQ ID NO: 5 RASSEQ ID NO: 6 QQTDEDPYT SEQ ID NO: 7 GFSLTTYG SEQ ID NO: 8 MWPGGRTSEQ ID NO: 9 VRGDYEYDYYAMDY SEQ ID NO: 10 SSVSY SEQ ID NO: 11 ATSSEQ ID NO: 12 HQWS SNPYT Humanised antibody 2.8.6 VH regionSEQ ID NO: 13 QVTLKESGPALVKPTQTLTLTCTVSGFSLTTYGVHWIRQPPGKALEWLGVMWPGGRTSYNPSLKSRLTITKDNSKSQVVLTMTNMDPVDTATYYCVRGDYEYDYYAMDYWG QGTLVTVSSHumanised antibody 2.8.6 VL region SEQ ID NO: 14EIVLTQSPATLSLSPGERATLSCRASSSVSYMHWYQQKPGQAPRPLIYATSNRATGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCHQWSSNPYTFGQGTKLEIKMouse Ab 2.8.6 VL region SEQ ID NO: 15QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCHQWSSNPYTFGGGTKLEIK SEQ ID NO: 16caaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagttcaagtgtaagttacatgcactggtaccagcagaagccaggatcctcccccaaaccctggatttatgccacatccaacctggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagaatggaggctgaagatgctgccacttattactgccaccagtggagtagtaacccgtacacgttcggaggggggaccaagctggaaataaaacSEQ ID NO: 17 = Mouse Ab 2.8.6 VH regionQVQLKESGPGLVAPSQSLSITCTVSGFSLTTYGVHWVRQSPGKGLEWLGVMWPGGRTSYNPAPMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCVRGDYEYDYYAMDYW GQGTSVTVSSSEQ ID NO: 18caggtgcagctgaaggagtctggacctggcctggtggcgccctcacagagcctgtccatcacttgcactgtctctgggttttcattaaccacctatggtgtacactgggttcgccagtctccaggaaagggtctggagtggctgggagtaatgtggcctggtggaagaacaagttataatccggctcccatgtccagactgagcatcagcaaagacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacggccatgtactactgtgtcagaggggactatgaatacgattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctcag Mouse Ab 11.5.1 VL region SEQ ID NO: 19DIVLTQSPASLAVSLGQRATISCRASETIDSYGDSLMHWYQQKAGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQTDEDPYTFGGGTKLEIK SEQ ID NO: 20gacattgtgctgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatatcctgcagagccagtgaaactattgatagttatggcgatagtbaatgcactggtaccagcagaaagcaggacagccacccaaactcctcatctatcgtgcatccaacctagaatctgggatccctgccaggttcagtggcagtgggtctcggacagacttcaccctcaccattaatcctgtggaggctgatgatgttgcaacctattactgtcagcaaactgatgaggatccgtacacgttcggaggggggaccaagctggaaataaaaMouse Ab 11.5.1 VH region SEQ ID NO: 21EVQLQESGPSLVKPSQTLSLTCSVTGDSITSAYWNVVIRKFPGNKLEYMGYISYSGSTYFNPSLKSRISITRNTSKNQYYLQLNSVTTEDTATYYCARSHYYGYYFDYWGHGTTLTVS SSEQ ID NO: 22gaggtgcagcttcaggagtcaggacctagcctcgtgaaaccttctcagactctgtccctcacctgttctgtcactggcgactccatcaccagtgcttactggaactggatccggaaattcccagggaataaacttgagtacatggggtacataagctacagtggtagcacttacttcaatccatctctcaaaagtcgaatctccatcactcgaaacacatccaagaaccagtactacctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagatctcattactacggctactactttgactactggggccatggcaccactctcacagtctcctcaHuman (Homo sapiens) BTLA polypeptide. Positions 1-30 is signal sequence,31-151 is extracellular region, 152-178 is transmembrane region and 179 to end is intracellularregion SEQ ID NO: 23MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCDVQLYIKRQSEHSILAGDPFELECPVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEPVLPNDNGSYRCSANFQSNLIESHSTTLYVTDVKSASERPSKDEMASRPWLLYRLLPLGGLPLLITTCFCLFCCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRScynomolgus monkey (Macaca fascicularis) BTLA polypeptide. SEQ ID NO: 24MKTLPAMLGSGRLFWVVFLIPYLDIWNIHGKESCDVQLYIKRQSYHSIFAGDPFKLECPVKYCAHRPQVTWCKLNGTTCVKLEGRHTSWKQEKNLSFFILHFEPVLPSDNGSYRCSANFLSAIIESHSTTLYVTDVKSASERPSKDEMASRPWLLYSLLPLGGLPLLITTCFCLFCFLRRHQGKQNELSDTTGREITLVDVPFKSEQTEASTRQNSQVLLSETGIYDNEPDFCFRMQEGSEVYSNPCLEENKPGIIYASLNHSIIGLNSRQARNVKEAPTEYASICVRS

TABLE 4 Exemplary CDR Sequences SEQ ID Amino Acid  SEQ ID Amino Acid NOs Sequences NOs Sequences 31 SYGIS 114 WQGTHFPQT 32 EIYPRSGNTYYNEKFKG117 TYYGSSQYYFDY 33 NYGSSYPFAY 121 DYYIN 34 SASSSVSSSYLH 122RIYPGSGNTYYNEKFKG 35 RTSNLAS 123 GYGNSDY 36 QQWSGYPFT 124 RASQSIGTRIH 37DYYMN 125 YASESIS 38 DINPNNGGTSYNQKFKG 126 QQSNSWPYT 39 WRQLRSDY 127SYAIR 40 LASQTIGTWLA 128 EIYPRSGNTYYNENFKG 41 AATSLAD 129 SGGASYTMDY 42QQLYSTPLT 133 SYGLI 43 SYWMH 134 EIYPRSGSTYYNEWFKG 44 MIHPNNGIPNYNEKFKS135 RRGTGDGFDY 45 EGYYGSEGYFDV 136 SASQGISNYLN 46 SASSSISYIH 137 YTSSLHS47 DTSKLAS 138 QQYIELPFT 48 HQRSTYPYT 139 DYYMH 56 MIHPNSGSTNYNEKFKS 140YIYPNNGGNGYNQKFKG 57 KRGGLGDY 141 GDYYGSLRLTFAY 58 RASKSVSTSGYSYMH 142KSSQSLLYSSNQKNYLA 59 LASNLES 143 WASTRES 60 QHSRELPYT 144 QQYYSYPLT 61SSWMN 145 TYGVS 62 RIYPGDGDTNYNGKFKG 146 WINTYSGVPTYADDFKG 63 RGYGYLAY147 VTTILHWYFDV 64 KASQDVSTAVA 148 RASQEISGYLS 65 SASYRYT 149 AASTLDS 66QQHYSTPYT 150 LQYASYPFT 69 GYGSSYGFAY 159 RRGAGDGFDY 72 QQWSGYPWT 162QQYSKLPFT 73 SGYYWN 169 DHTIH 74 YISYDGSNNYNPSLKN 170 YIYPRDGSTKYNEKFKG75 IYGNYYAMDY 171 SNWNFDY 76 SASSSVSYMH 172 KASQDVGTAVA 78 QQWSSNPPT 173WASTRRT 79 DYYMI 174 QQYSSYPLT 80 NINPNNGGTTYNQKFKG 180 QQHYSTPWT 81GGLRPLYFDY 182 EIYPRSGTTYYNEKFKG 82 KASENVDTYVS 183 RISSGSGVDY 83GASNRYT 186 QQYSELPWT 84 GQSYSYPLT 187 SGYDWH 85 NTYMH 188YISYSGSTNYNPSLKS 86 RIDPANGNTKYDPKFQG 189 GTPVVAEDYFDY 87 TYYGSSQHYFDY190 RSSTGAVTTSNYAN 88 KSSQSLLDSDGKTYLN 191 ATNNRAP 89 LVSKLDS 192ALWYSNHLV 90 WQDTHFPQT 193 TYGVH 92 RIYPGDGDANYNGKFKG 194VMWPGGRTSYNPAPMS 93 EGHYYGSGYRWYLDV 195 GDYEYDYYAMDY 94 RASENIYSNLA 196RASSSVSYMH 95 AATNLAD 197 ATSNLAS 96 QHFRGAPFT 199 SAYWN 97 DYEIH 200YISYSGSTYFNPSLKS 98 PIDPDTGNTAYNQNLKG 201 SHYYGYYFDY 99 GGYDSDWGFAY 202RASETIDSYGDSLMH 100 RSSKSLLHSNGNTFLF 203 RASNLES 101 RMSDLAS 205 SYGMS102 MQHLEYPFT 206 SIRSDGNTYYPDSVKG 103 DYYLN 207 GGYYGSSPYY 104LIDPYNGGSSCNQKFKG 209 WASTRDS 105 GNAMDY 210 QQYYNYLT 107 WASTRHT 211SGYSWH 108 QQHYIIPYM 212 YIHYSGSTNYNPSLKS 109 NTYMY 213 GPHRYDGVWFAY 110RIDPANGNTKYAPKFQG 214 SASSSISSNYLH 111 LYYGSSYDYFDY 216 QQGTNIPLT 386EIYPRSGNTYYAQKFQG 388 EIYPRSGQTYYAQSFQG 387 SIRSEGQTYYPDSVKG

TABLE 5 Exemplary Primary VH and VL Sequences SEQ ID NOsAmino Acid Sequences 301QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYPRSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARNYG SSYPFAYWGQGTLVTVSA302 EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNVVVKQSHGKSLEWIGDINPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARWR QLRSDYWGQGTTLTVSS 303QVQLQQPGAELVKPRASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGMIHPNNGIPNYNEKFKSKATLTVDKSSTTAYMQLSSLTSEDSAVYHCAREGYYGSEGYFDVWGTGTTVTVSS 305QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARKR GGLGDYWGQGTSVTVSS306  QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARR GYGYLAYWGQGTLVTVSA307 QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYPRSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARGYG SSYGFAYWGQGTLVTVSA308 DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCASIYGN YYAMDYWGQGTSVTVSS309 EVQLQQSGPELVQPGASVKISCKASGYTFTDYYMIWVKQSHGKSLEWIGNINPNNGGTTYNQKFKGKATLTVDKSSSTAYMGLPSLTSEDSAVYYCARGGL RPLYFDYWGQGTTLTVSS310 EVQLQQSVAELVRPGASVKLSCTASGFNIKNTYMHWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYVQLSSLTSEDTAIYYCALTYY GSSQHYFDYWGQGTTLTVSS311 QIQLQQSGPELVKPGASVKISCKASGYAFSSSWMNVVVKKRPGKGLEWIGRIYPGDGDANYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAGEGHYYGSGYRWYLDVWGTGTTVTVSS 312QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEIHWVKQTLVHGLEWIGPIDPDTGNTAYNQNLKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGGY DSDWGFAYWGQGTLVTVSA313 EVQLQQSGPVLVKPGASVKMSCKASGYTFTDYYLNWVKQSHGKSLEWIGLIDPYNGGSSCNQKFKGKATLTVDKSSSTAYMDLNSLTSEDSAVYYCARG NAMDYWGQGTSVTVSS 314EVQLQQSVAELVRPGASVKLSCTASGFNIKNTYMYVVVKQRPEQGLEWIGRIDPANGNTKYAPKFQGKATITADTSSNTAYLQLSSLTSEDTAIYYCALLYYG SSYDYFDYWGQGTTLTVSS315  EVQLQQSVAELVRPGASVKLSCTASGFNIKNTYMHWVKQRPEQGLEWIGRIDPANGNTKYAPKFQGKATITADTSSNTAYLQLSSLTSEDTAIYYCALTYYG SSQYYFDYWGQGTTLTVSS316 QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYPGSGNTYYNEKFKGKATLTAEKSSSTAYMQLSSLTSEDSAVYFCARGYG NSDYWGQGTTLTVSS 317QVQLQQSGAELARPGASVRLSCKASGYTFTSYAIRWVKQRTGQGLEWIGEIYPRSGNTYYNENFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARSGG ASYTMDYWGQGTSVTVSS318 QVQLQQSGAELARPGASVRLSCKASGYTFTSYGLIWLKQRTGQGLEWIGEIYPRSGSTYYNEWFKGKATLTADKSSNTAFMELRSLTSEDSAVYFCARRRG TGDGFDYWGQGTILTVSS319 EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMHWVKQSHGKSLEWIGYIYPNNGGNGYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAIGDYYGSLRLTFAYWGQGTLVTVSA 320 QIQLVQSGPELKKPGETVKISCKASGYTFTTYGVSWVKQAPGKVLKWMGWINTYSGVPTYADDFKGRFAFSLETSASTAYLQISNLKNEDTATYFCAPVT TILHWYFDVWGTGTTVTVSS321 QVQLQQSGAELARPGASVRLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYPRSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARNYG SSYPFAYWGQGTLVTVSA322 QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYPRSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARRRG AGDGFDYWGQGTTLTVSS324 QDQLQQSDAELVKPGASVKISCKVSGYTFTDHTIHWMKQRPEQGLEWIGYIYPRDGSTKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCASSN WNFDYWGQGTTLTVSS 326QVQLQQSGAELARPGASVKLPCKASGYTFTSYGISWVKQRTGQGLEWIGEIYPRSGTTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARRISS GSGVDYVVGQGTTLTVSS327 DVQLQESGPGMVKPSQSLSLTCTVTGYSITSGYDWHWIRHFPGNKLEWMGYISYSGSTNYNPSLKSRISITHDTSKNHFFLKLNSVTTEDTATYYCARGTPV VAEDYFDYWGQGTTLTVSS330 EVKLVESGGGLVKPGGSLKLSCAASGFTLSSYGMSWVRQIPEKRLEWVASIRSDGNTYYPDSVKGRFIISRDNARNILYLQMSSLRSEDTAMYYCTRGGYYG SSPYYWGQGTTLTVSS 331DVQLQESGPDLVKPSQSLSVTCTVTGYSITSGYSWHWIRQFPGNKLEWMGYIHYSGSTNYNPSLKSRISITRDTSKNQFFLQLSSVTTEDTATYYCASGPHRY DGVWFAYWGQGTLVTVSS351 ENVLTQSPAIMAASLGQKVTMTCSASSSVSSSYLHWYQQKSGASPKPLIHRTSNLASGVPARFSGSGSGTSYSLTISSVEAEDDATYYCQQWSGYPFTFGGG TKLEIK 352DIQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQLLIYAATSLADGVPSRFSGSGSGTKFSFKISSLQAEDFVSYYCQQLYSTPLTFGAGTKL ELK 353QIVLTQSPAIMSASPGEKVTMTCSASSSISYIHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSTYPYTFGGGTKL EIK 355DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPYTFGG GTKLEIK 356DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPYTFGGG TKLEIK 357ENVLTQSPAIMAASLGQKVTMTCSASSSVSSSYLHWYQQKSGASPKPLIHRTSNLASGVPARFSGSGSGTSYSLTISSVEAEDDATYYCQQWSGYPWTFGGG TKLEIK 358QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGSGTK LEIK 359NIVMTQSPKSMSMSVGERVTLSCKASENVDTYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQSYSYPLTFGAG TKLELI 360DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQDTHFPQTF GGGTKLEIK 361DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFRGAPFTFGSGTK LEIK 362DIVMTQATPSVPVTPGESVSISCRSSKSLLHSNGNTFLFWFLQRPGQSPQLLIYRMSDLASGVPDRFSGSGSGTAFTLRISRVEAEDVGIYYCMQHLEYPFTFG SGTKLEIK 363DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQEKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYILNISSVQAEDLALYYCQQHYIIPYMFGGGT KLEIK 364DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTF GGGTKLEIK 366DILLTQSPAILSVSPGERVSFSCRASQSIGTRIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPYTFGGGTKLEIK 367ENVLTQSPAIMAASLGQKVTMTCSASSSVSSSYLHWYQQKSGASPKPLIHRTSNLASGVPARFSGSGSGTSYSLTISSVEAEDDATYYCQQWSGYPFTFGSGT KLEIK 368DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNVVYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYIELPFTFGSGTKLEI K 369DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYVVASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYP LTFGAGTKLELK 370DIQMTQSPSSLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFRGSRSGSDYSLTISSLESEDFADYYCLQYASYPFTFGSGTKLEI K 372DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNVVYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPFTFGSGTKLE IK 374DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRRTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPLTFGAGT KLELK 375DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGG TKLEIK 376DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNVVYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSELPWTFGGGTKL EIK 377QAVVTQESALSTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGATNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHLVFGG GTKLTVLG 378DIVMTQSPLSLPVTPGEPASISCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYVVASTRESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYNYLTF GGGTKVEIK 380DIVMSQSPSSLPVSVGEKISMTCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRDSGVPDRFIGSGSGTDFTLTINSVKAEDLAVYYCQQYYNYLT FGAGTKLELK 381EIVLTQSPTTMAASPGEKITITCSASSSISSNYLHWYQQKPGFSPKWYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGTNIPLTFGAGTKL EIK 382QVQLVQSGAELKKPGASVKVSCKASGYTFTSYAIRWVRQATGQGLEWMGEIYPRSGNTYYAQKFQGRATLTADKSISTAYMELSSLRSEDTAVYFCARSG GASYTMDYWGQGTTVTVSS383 ENVLTQSPATLSLSPGERATLSCSASSSVSSSYLHWYQQKPGQSPRPLIHRTSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSGYPFTFGSGTKL EIK 384EVQLVESGGGLVKPGGSLRLSCAASGFTLSSYGMSWVRQAPGKGLEWVASIRSDGNTYYPDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAVYYCTRGGY YGSSPYYWGQGTTVTVSS385 DIVMTQSPLSLPVTPGEPASISCKSSQSLLYSSNQKNYLAWYQQKPGQSPQLLIYVVASTRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYNYLTF GGGTKVEIK 389QVQLVQSGAELKKPGASVKVSCKASGYTFTSYAIRWVRQATGQGLEWMGEIYPRSGQTYYAQSFQGRATLTADKSTSTAYMELSSLRSEDTAVYFCARSG GASYTMDYWGQGTTVTVSS390 EVQLVESGGGLVKPGGSLRLSCAASGFTLSSYGMSWVRQAPGKGLEWVASIRSEGQTYYPDSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCTRGGY YGSSPYYWGQGTTVTVSS

1-95. (canceled)
 96. A method of modulating an immune response in asubject, comprising administering to the subject an antibody or anantigen-binding fragment thereof that specifically binds B and TLymphocyte Attenuator (BTLA), wherein the antibody or antigen-bindingfragment thereof: (i) comprises a heavy chain and a light chain; (ii)agonizes BTLA expressed on the surface of an immune cell; and (iii)binds a residue of BTLA selected from the group consisting of: D52, P53,E55, E57, E83, Q86, E103, L106 and E92.
 97. The method of claim 96,wherein the antibody or antigen-binding fragment thereof does notinhibit binding of BTLA to herpes virus entry mediator (HVEM).
 98. Themethod of claim 96, wherein the antibody or antigen-binding fragmentthereof binds human BTLA at a KD of less than 10 nM, as determined bysurface plasmon resonance at 37° C.
 99. The method of claim 96, whereinthe antibody or antigen-binding fragment thereof binds human BTLA at aKD of less than 2 nM, as determined by surface plasmon resonance at 37°C.
 100. The method of claim 96, wherein the antibody or antigen-bindingfragment thereof binds cynomolgus BTLA at a KD of less than 20 nM, asdetermined by surface plasmon resonance at 37° C.
 101. The method ofclaim 96, wherein the antibody or antigen-binding fragment thereofinhibits proliferation of T cells in the subject.
 102. The method ofclaim 96, wherein: (a) the light chain comprises a light chain variableregion that comprises a CDR that has an amino acid sequence as set forthin any of SEQ ID NO: 142, SEQ ID NO: 143, or SEQ ID NO: 210, with from 0to 3 amino acid modifications; and (b) the heavy chain comprises a heavychain variable region that comprises a CDR that has an amino acidsequence as set forth in SEQ ID NO: 205, SEQ ID NO: 387, and SEQ ID NO:207, with from 0 to 3 amino acid modifications.
 103. The method of claim96, wherein the light chain variable region comprises an amino acidsequence having at least 90% identity to the amino acid sequence as setforth in SEQ ID NO:
 378. 104. The method of claim 103, wherein the heavychain variable region comprises an amino acid sequence having at least90% identity to the amino acid sequence as set forth in SEQ ID NO: 390.105. The method of claim 96, wherein: (a) the light chain comprises alight chain variable region that comprises three CDRs: CDRL1, CDRL2, andCDRL3, wherein (a) CDRL1 has the amino acid sequence as set forth in SEQID NO: 142, with 0 to 1 amino acid modification; (b) CDRL2 has the aminoacid sequence as set forth in SEQ ID NO: 143, with 0 to 1 amino acidmodification; and (c) CDRL3 has the amino acid sequence as set forth inSEQ ID NO: 210, with 0 to 1 amino acid modification; and (b) the heavychain comprises a heavy chain variable region comprising three CDRs:CDRH1, CDRH2, and CDRH3, wherein (a) CDRH1 has the amino acid sequenceas set forth in SEQ ID NO: 205, with 0 to 1 amino acid modification; (b)CDRH2 has the amino acid sequence as set forth in SEQ ID NO: 387, with 0to 2 amino acid modifications; and (c) CDRH3 has the amino acid sequenceas set forth in SEQ ID NO: 207, with 0 to 1 amino acid modification.106. The method of 105, wherein CDRL2 has no amino acid modifications ascompared to SEQ ID NO:
 143. 107. The method of 105, wherein CDRH2 has anamino acid modification at position 5, position 7, or both, numberedaccording to SEQ ID NO:
 387. 108. The method of 107, wherein CDRL2 hasno amino acid modifications as compared to SEQ ID NO:
 143. 109. Themethod of 105, wherein the light chain variable region comprises theamino acid sequence as set forth in SEQ ID NO:
 378. 110. The method ofclaim 96, wherein the antibody or antigen-binding fragment thereoffurther comprises a domain that binds to an Fc receptor.
 111. The methodof claim 110, wherein said Fc receptor is FcγRIIB
 112. The method ofclaim 96, wherein the antibody or antigen-binding fragment thereof is ahumanized antibody.
 113. The method of claim 96, wherein the antibody orantigen-binding fragment thereof is a monoclonal antibody.
 114. Themethod of claim 96, wherein the heavy chain or light chain also comprisea constant region.
 115. The method of claim 96, wherein the heavy chainand light chain are connected by a flexible linker to form asingle-chain antibody.
 116. An antibody or an antigen-binding fragmentthereof that specifically binds BTLA, comprising a heavy chain and alight chain, wherein the heavy chain comprises a heavy chain variableregion comprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein (1) CDRH1,CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 31,SEQ ID NO: 32, and SEQ ID NO: 33, respectively, with from 0 to 3 aminoacid modification, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO:36, respectively, with from 0 to 3 amino acid modifications; (2) CDRH1,CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 37,SEQ ID NO: 38, and SEQ ID NO: 39, respectively, with from 0 to 3 aminoacid modification, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO:42, respectively, with from 0 to 3 amino acid modifications; (3) CDRH1,CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 43,SEQ ID NO: 44, and SEQ ID NO: 45, respectively, with from 0 to 3 aminoacid modification, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 6, SEQ ID NO: 47, and SEQ ID NO: 48,respectively, with from 0 to 3 amino acid modifications; (4) CDRH1,CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 43,SEQ ID NO: 56, and SEQ ID NO: 57, respectively, with from 0 to 3 aminoacid modification, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO:60, respectively, with from 0 to 3 amino acid modifications; (5) CDRH1,CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 61,SEQ ID NO: 62, and SEQ ID NO: 63, respectively, with from 0 to 3 aminoacid modification, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO:66, respectively, with from 0 to 3 amino acid modifications; (6) CDRH1,CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 31,SEQ ID NO: 32, and SEQ ID NO: 69, respectively, with from 0 to 3 aminoacid modification, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO:72, respectively, with from 0 to 3 amino acid modifications; (7) CDRH1,CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 73,SEQ ID NO: 74, and SEQ ID NO: 75, respectively, with from 0 to 3 aminoacid modification, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 76, SEQ ID NO: 47, and SEQ ID NO:78, respectively, with from 0 to 3 amino acid modifications; (8) CDRH1,CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 79,SEQ ID NO: 80, and SEQ ID NO: 81, respectively, with from 0 to 3 aminoacid modification, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO:84, respectively, with from 0 to 3 amino acid modifications; (9) CDRH1,CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 85,SEQ ID NO: 86, and SEQ ID NO: 87, respectively, with from 0 to 3 aminoacid modification, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO:90, respectively, with from 0 to 3 amino acid modifications; (10) CDRH1,CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 61,SEQ ID NO: 92, and SEQ ID NO: 93, respectively, with from 0 to 3 aminoacid modification, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO:96, respectively, with from 0 to 3 amino acid modifications; (11) CDRH1,CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 97,SEQ ID NO: 98, and SEQ ID NO: 99, respectively, with from 0 to 3 aminoacid modification, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO:102, respectively, with from 0 to 3 amino acid modifications; (12)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively, with from 0to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 64, SEQ ID NO: 107, and SEQ IDNO: 108, respectively, with from 0 to 3 amino acid modifications; (13)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, respectively, with from 0to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ IDNO: 114, respectively, with from 0 to 3 amino acid modifications; (14)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 85, SEQ ID NO: 110, and SEQ ID NO: 117, respectively, with from 0 to3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 88, SEQ ID NO: 89, and SEQ IDNO: 114, respectively, with from 0 to 3 amino acid modifications; (15)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, respectively, with from 0to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 124, SEQ ID NO: 125, and SEQ IDNO: 126, respectively, with from 0 to 3 amino acid modifications; (16)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, respectively, with from 0to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ IDNO: 36, respectively, with from 0 to 3 amino acid modifications; (17)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 133, SEQ ID NO: 134, and SEQ ID NO: 135, respectively, with from 0to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 136, SEQ ID NO: 137, and SEQ IDNO: 138, respectively, with from 0 to 3 amino acid modifications; (18)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 139, SEQ ID NO: 140, and SEQ ID NO: 141, respectively, with from 0to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 142, SEQ ID NO: 143, and SEQ IDNO: 144, respectively, with from 0 to 3 amino acid modifications; (19)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 145, SEQ ID NO: 146, and SEQ ID NO: 147, respectively, with from 0to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 148, SEQ ID NO: 149, and SEQ IDNO: 150, respectively, with from 0 to 3 amino acid modifications; (20)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively, with from 0 to 3amino acid modification, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO:150, respectively, with from 0 to 3 amino acid modifications; (21)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 31, SEQ ID NO: 32, and SEQ ID NO: 159, respectively, with from 0 to3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 136, SEQ ID NO: 137, and SEQ IDNO: 162, respectively, with from 0 to 3 amino acid modifications; (22)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 169, SEQ ID NO: 170, and SEQ ID NO: 171, respectively, with from 0to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 172, SEQ ID NO: 173, and SEQ IDNO: 174, respectively, with from 0 to 3 amino acid modifications; (23)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 61, SEQ ID NO: 62, and SEQ ID NO: 63, respectively, with from 0 to 3amino acid modification, and CDRL1, CDRL2, and CDRL3 have an amino acidsequence as set forth in SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO:180, respectively, with from 0 to 3 amino acid modifications; (24)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 31, SEQ ID NO: 182, and SEQ ID NO: 183, respectively, with from 0 to3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 136, SEQ ID NO: 137, and SEQ IDNO: 186, respectively, with from 0 to 3 amino acid modifications; (25)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 187, SEQ ID NO: 188, and SEQ ID NO: 189, respectively, with from 0to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 190, SEQ ID NO: 191, and SEQ IDNO: 192, respectively, with from 0 to 3 amino acid modifications; (26)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 193, SEQ ID NO: 194, and SEQ ID NO: 195, respectively, with from 0to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 196, SEQ ID NO: 197, and SEQ IDNO: 12, respectively, with from 0 to 3 amino acid modifications; (27)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 199, SEQ ID NO: 200, and SEQ ID NO: 201, respectively, with from 0to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 202, SEQ ID NO: 203, and SEQ IDNO: 6, respectively, with from 0 to 3 amino acid modifications; (28)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 211, SEQ ID NO: 212, and SEQ ID NO: 213, respectively, with from 0to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 214, SEQ ID NO: 35, and SEQ IDNO: 216, respectively, with from 0 to 3 amino acid modification; (29)CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ IDNO: 127, SEQ ID NO: 386, and SEQ ID NO: 129, respectively, with from 0to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an aminoacid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ IDNO: 36, respectively, with from 0 to 3 amino acid modification; ; or(30) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQID NO: 127, SEQ ID NO: 388, and SEQ ID NO: 129, respectively, with from0 to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have anamino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35, andSEQ ID NO: 36, respectively, with from 0 to 3 amino acid modification.